Antibodies that bind zika virus envelope protein and uses thereof

ABSTRACT

Isolated monoclonal antibodies which bind to Zika virus envelope protein and related antibody-based compositions and molecules are disclosed. Also disclosed are therapeutic and diagnostic methods for using the antibodies.

RELATED APPLICATIONS

This application claims the benefits of the priority date of U.S.Provisional Application No. 62/408,020, which was filed on Oct. 13,2016. The contents of this provisional application is herebyincorporated by reference in its entirety.

BACKGROUND

Zika virus (ZIKV) is a vector-borne arbovirus transmitted by Aedesaegypti mosquito. ZIKV infection typically causes mild symptoms,including fever, rash, joint pain, and/or conjunctivitis. However,recent research indicates a link between infection and microcephaly innewborn babies, along with a link between infection and Guillain-barresyndrome in adults. There is an urgent need for effective countermeasures as there are no approved vaccines or therapies against ZIKV.

Little is known about the virus, structure, or biology of ZIKV. ZIKV isa member of the virus family Flaviviridae, which includes Dengue (DV),West Nile, Japanese Encephalitis, Tick-born Encephalitis, and YellowFever virus. The envelope (E) protein of flavivirus mediates host cellentry and immune evasion. The E protein consists of three structural andfunctional domains. Antibodies against E protein domain III (E-DIII)have shown prophylactic and therapeutic effects in animal modelsinfected with DV (Robinson, L., et al., Cell Vol. 162: 493-504, 2015).Therefore, antibody-based agents against ZIKV envelope protein provide apromising option for combating ZIKV outbreaks in humans.

SUMMARY

The present disclosure pertains to antibodies directed towards Zikavirus envelope protein (EP). A systematic analysis of the Zika virussurface guided by the residue interatomic interactions network (or SIN)led to the identification of fusion loop epitope proximal (FLEP) regionas being structurally constrained. A structure based computationalapproach yielded a set of promising scaffolds with potential to interactwith the FLEP region. Following this, an anti-TDRD3 (Tudor DomainContaining 3) antibody was investigated due to its ability to interfacewith the FLEP region. A framework to compute the inter-residue atomicinteraction between interacting amino acid pairs of the antigen-antibodyinterface was utilized, and interactions were rendered in a 2D graphformat to analyze the connectivity network. Mutations in the CDRs and/orframework regions that contributed to more favorable contacts, asevaluated by the structural analysis and connectivity network, wereidentified and various amino acid residues which potentially mediate newor improved contacts were analyzed to identify CDR and/or frameworkmutations that would result in binding to Zika virus EP. The identifiedantibodies were found to treat and prevent Zika virus infection in asubject, as well as prevent vertical infection and fetal mortality in apregnant subject.

Accordingly, the present disclosure relates to antibodies that bind Zikavirus EP. Also provided herein are host cells and methods for treatingZika virus with these antibodies.

In some aspects, the isolated monoclonal antibody which specificallybinds Zika virus envelope protein, or antigen binding portion thereof,comprises heavy and light chain CDRs, wherein

(i) heavy chain CDR1 comprises GFX₁FSTY, wherein X₁ may or may not bepresent, and if present is a polar amino acid residue;

(ii) heavy chain CDR2 comprises X₂GEGDS, wherein X₂ is a polar aminoacid residue;

(iii) heavy chain CDR3 comprises GYX₃NFYYYYTMDX₄, wherein X₃ is a polaramino acid residue and X₄ is a nonpolar amino acid residue;

(iv) light chain CDR1 comprises RAX₅QSIX₆TFLA, wherein X₅ is a polaramino acid residue and X₆ is a polar amino acid residue or a hydrophobicamino acid residue;

(v) light chain CDR2 comprises DASTX₇AX₈, wherein X₇ and X₈ are polaramino acids; and

(vi) light chain CDR3 comprises QQRYNWPPYX₉, wherein X₉ is a polar aminoacid.

In other aspects, provided herein is an isolated monoclonal antibody, orantigen binding portion thereof, comprising heavy and light chain CDRswherein

(i) heavy chain CDR1 comprises GFX₁FSTY, wherein X₁ is selected from Sand T;

(ii) heavy chain CDR2 comprises X₂GEGDS, wherein X₂ is selected from Sand T;

(iii) heavy chain CDR3 comprises GYX₃NFYYYYTMDX₄, wherein X₃ is selectedfrom S and T and X₄ is selected from A and V;

(iv) light chain CDR1 comprises RAX₅QSIX₆TFLA, wherein X₅ is selectedfrom S and T and X₆ is selected from S and V;

(v) light chain CDR2 comprises DASTX₇AX₈, wherein X₇ is selected from Rand N and X₈ is selected from S and T; and

(vi) light chain CDR3 comprises QQRYNWPPYX₉, wherein X₉ is selected fromS and T.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFX₁FSTY, wherein X₁ is not present;

(ii) heavy chain CDR2 comprising X₂GEGDS, wherein X₂ is selected from Sand T;

(iii) heavy chain CDR3 comprising GYX₃NFYYYYTMDX₄, wherein X₃ isselected from S and T and X₄ is selected from A and V;

(iv) light chain CDR1 comprising RAX₅QSIX₆TFLA, wherein X₅ is selectedfrom S and T and X₆ is selected from S and V;

(v) light chain CDR2 comprising DASTX₇AX₈, wherein X₇ is selected from Rand N and X₈ is selected from S and T; and

(vi) light chain CDR3 comprising QQRYNWPPYX₉, wherein X₉ is selectedfrom S and T.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises,

(i) heavy chain CDR1 comprising GFSFSTY;

(ii) heavy chain CDR2 comprising SGEGDS; and

(iii) heavy chain CDR3 comprising GYSNFYYYYTMDA.

In other aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFSFSTY;

(ii) heavy chain CDR2 comprising TGEGDS; and

(iii) heavy chain CDR3 comprising GYSNFYYYYTMDA.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFTFSTY;

(ii) heavy chain CDR2 comprising TGEGDS; and

(iii) heavy chain CDR3 comprising GYSNFYYYYTMDV.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFFSTY;

(ii) heavy chain CDR2 comprising TGEGDS; and

(iii) heavy chain CDR3 comprising GYTNFYYYYTMDA.

In other aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFSFSTY;

(ii) heavy chain CDR2 comprising TGEGDS; and

(iii) heavy chain CDR3 comprising GYTNFYYYYTMDA.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(iv) light chain CDR1 comprising RATQSISTFLA;

(v) light chain CDR2 comprising DASTRAS; and

(vi) light chain CDR3 comprising QQRYNWPPYS.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(iv) light chain CDR1 comprising RASQSISTFLA;

(v) light chain CDR2 comprising DASTRAT; and

(vi) light chain CDR3 comprising QQRYNWPPYT.

In other aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(iv) light chain CDR1 comprising RATQSIVTFLA;

(v) light chain CDR2 comprising DASTNAS; and

(vi) light chain CDR3 comprising QQRYNWPPYS.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFSFSTY;

(ii) heavy chain CDR2 comprising SGEGDS;

(iii) heavy chain CDR3 comprising GYSNFYYYYTMDA;

(iv) light chain CDR1 comprising RATQSISTFLA;

(v) light chain CDR2 comprising DASTRAS; and

(vi) light chain CDR3 comprising QQRYNWPPYS.

In other aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFSFSTY;

(ii) heavy chain CDR2 comprising SGEGDS;

(iii) heavy chain CDR3 comprising GYSNFYYYYTMDA;

(iv) light chain CDR1 comprising RATQSIVTFLA;

(v) light chain CDR2 comprising DASTNAS; and

(vi) light chain CDR3 comprising QQRYNWPPYS.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFSFSTY;

(ii) heavy chain CDR2 comprising TGEGDS;

(iii) heavy chain CDR3 comprising GYSNFYYYYTMDA;

(iv) light chain CDR1 comprising RATQSISTFLA;

(v) light chain CDR2 comprising DASTRAS; and

(vi) light chain CDR3 comprising QQRYNWPPYS.

In other aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises

(i) heavy chain CDR1 comprising GFTFSTY;

(ii) heavy chain CDR2 comprising TGEGDS;

(iii) heavy chain CDR3 comprising GYSNFYYYYTMDV;

(iv) light chain CDR1 comprising RASQSISTFLA;

(v) light chain CDR2 comprising DASTRAT; and

(vi) light chain CDR3 comprising QQRYNWPPYT.

In some aspects, the disclosure provides an isolated monoclonal antibodywhich specifically binds Zika virus envelope protein, or antigen bindingportion thereof, comprising a heavy chain variable region as set forthin SEQ ID NO: 4, wherein

(i) heavy chain CDR1 comprises an amino acid substitution or deletion atN28, and amino acid substitutions at L29, S31, S32;

(ii) heavy chain CDR2 comprises amino acid substitutions at S52, S52A,S53, Y54, G55;

(iii) heavy chain CDR3 comprises an amino acid deletion at S99 and aminoacid substitutions at K100, K100A, P100B, Y100C, F100D, S100E, G100F,W100G, A100H, and Y102; and

wherein the heavy chain variable region comprises at least one aminoacid deletion at R94, and at CDR3 residues T95, V96, and R97, andcombinations thereof, numbering according to Chothia. In some aspects,the heavy chain variable region further comprises at least one aminoacid substitution at V5, Y33, A49, S50, T57, A71, T73, A78, S82B, L82C,A93, L108 and combinations thereof, numbering according to Chothia. Insome aspects, the heavy chain variable region further comprises an aminoacid substitution at T68, numbering according to Chothia. In someaspects, the heavy chain variable region further comprises at least oneamino acid substitution at A23, W47, Y58, L80, Q81, A84, andcombinations thereof, numbering according to Chothia. In some aspects,the heavy chain variable region further comprises at least one aminoacid substitution at E1, A23, R38, W47, Y58, L80, Q81, A84, andcombinations thereof, numbering according to Chothia. In some aspects,the heavy chain variable region further comprises at least one aminoacid substitution at E1, A23, R38, W47, Y58, T68, L80, Q81, A84, andcombinations thereof, numbering according to Chothia.

In any of the foregoing aspects, the isolated monoclonal antibody, orantigen binding portion thereof, described herein, comprising a heavychain variable region as set forth in SEQ ID NO: 4, comprises

(i) heavy chain CDR1 comprising N28S or N28T, L29F, S31T, S32Y;

(ii) heavy chain CDR2 comprising S52T, S52AG, S53E, Y54G, G55D;

(iii) heavy chain CDR3 comprising K100Y, K100AS or K100AT, P100BN,Y100CF, F100DY, S100EY, G100FY, W100GY, A100HT, and Y102A or Y102V.

In some aspects, the heavy chain variable region comprises amino aciddeletions at R94, and at CDR3 residues T95, V96, and R97, numberingaccording to Chothia.

In any of the foregoing aspects, the isolated monoclonal antibody, orantigen binding portion thereof, described herein, comprises a lightchain variable region comprising an amino acid sequence set forth in SEQID NO: 5, wherein

(i) light chain CDR1 comprises amino acid substitutions at S26, V29,S31, A32, and V33;

(ii) light chain CDR2 comprises amino acid substitutions at S50, S53,L54, Y55 and optionally S56; and

(iii) light chain CDR3 comprises amino acid substitutions at H91, P93,F94, Y95, L95B, F96, and T97 and an amino acid deletion at G92.

In any of the foregoing aspects, the isolated monoclonal antibody, orantigen binding portion thereof, described herein, comprises

(i) light chain CDR1 comprising S26T, V29I, S31V, A32F, and V33L;

(ii) light chain CDR2 comprising S50D, S53T, L54R or L54N, Y55A and,optionally S56T; and

(ii) light chain CDR3 comprising amino acid substitutions at H91R, P93Y,F94N, Y95W, L95BP, F96Y, and T97S.

In any of the foregoing aspects, the light chain variable region furthercomprises at least one amino acid substitution at D1, Q3, M4, S9, A13,V15, D17, V19, 121, Y22, Q38, K42, K45, S60, Q79, T85, and combinationsthereof, numbering according to Chothia. In some aspects, the lightchain variable region further comprises at least one amino acidsubstitution at S10, V58, S76, S77, V104, and combinations thereof,numbering according to Chothia.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, which binds to Zika virusenvelope protein and comprises heavy and light chain variable regions,wherein the heavy and light chain amino acid sequences are selected fromthe group consisting of:

(a) SEQ ID NOs: 4 and 14, respectively;

(b) SEQ ID NOs: 4 and 15, respectively;

(c) SEQ ID NOs: 9 and 16, respectively;

(d) SEQ ID NOs: 4 and 16, respectively;

(e) SEQ ID NOs: 4 and 17, respectively;

(f) SEQ ID NOs: 8 and 14, respectively;

(g) SEQ ID NOs: 7 and 17, respectively;

(h) SEQ ID NOs: 6 and 15, respectively;

(i) SEQ ID NOs: 6 and 5, respectively;

(j) SEQ ID NOs: 7 and 5, respectively;

(k) SEQ ID NOs: 8 and 5, respectively;

(l) SEQ ID NOs: 9 and 5, respectively;

(m) SEQ ID NOs: 10 and 5, respectively;

(n) SEQ ID NOs: 11 and 5, respectively;

(o) SEQ ID NOs: 6 and 14, respectively;

(p) SEQ ID NOs: 6 and 16, respectively;

(q) SEQ ID NOs: 6 and 17, respectively;

(r) SEQ ID NOs: 7 and 14, respectively;

(s) SEQ ID NOs: 7 and 15, respectively;

(t) SEQ ID NOs: 7 and 16, respectively;

(u) SEQ ID NOs: 8 and 15, respectively;

(v) SEQ ID NOs: 8 and 16, respectively;

(w) SEQ ID NOs: 8 and 17, respectively;

(x) SEQ ID NOs: 9 and 14, respectively;

(y) SEQ ID NOs: 9 and 15, respectively;

(z) SEQ ID NOs: 9 and 17, respectively;

(aa) SEQ ID NOs: 10 and 14, respectively;

(bb) SEQ ID NOs: 10 and 15, respectively;

(cc) SEQ ID NOs: 10 and 16, respectively;

(dd) SEQ ID NOs: 10 and 17, respectively;

(ee) SEQ ID NOs: 11 and 14, respectively;

(ff) SEQ ID NOs: 11 and 15, respectively;

(gg) SEQ ID NOs: 11 and 16, respectively; and

(hh) SEQ ID NOs: 11 and 17, respectively.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, which binds to Zika virusenvelope protein, comprising heavy and light chain CDRs selected fromthe group consisting of:

(a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:20, 26 and 31, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:22, 28 and 33, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:20, 26 and 31, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:20, 26 and 31, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 28 and 32, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 27 and 32, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 27 and 32, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively;

(h) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 27 and 32, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 28 and 32, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively;

(j) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:22, 28 and 33, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively;

(k) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:23, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively;

(l) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively;

(m) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 27 and 32, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively;

(n) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 28 and 32, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively;

(o) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 28 and 32, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively;

(p) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:22, 28 and 33, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively;

(q) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:22, 28 and 33, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively;

(r) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:23, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively;

(s) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:23, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively;

(t) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:23, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively;

(u) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively;

(v) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively; and

(w) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:21, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, which binds to Zika virusenvelope protein and comprises heavy and light chain variable regions,wherein the heavy chain variable region comprises an amino acid sequencewhich is at least 90% identical to the amino acid sequence selected fromthe group consisting of SEQ ID NOs: 4, 6, 7, 8, 9, 10 and 11; andwherein the light chain variable region comprises an amino acid sequencewhich is at least 90% identical to the amino acid sequence selected fromthe group consisting of SEQ ID NOs: 5, 14, 15, 16, and 17, provided thatthe monoclonal antibody does not comprise SEQ ID NOs: 4 and 5.

In some aspects, the isolated monoclonal antibody, or antigen bindingportion thereof, described herein, comprises heavy chain and light chainsequences having at least 90% identity to the heavy and light chainamino acid sequences selected from the group consisting of:

(a) SEQ ID NOs: 4 and 14, respectively;

(b) SEQ ID NOs: 4 and 15, respectively;

(c) SEQ ID NOs: 9 and 16, respectively;

(d) SEQ ID NOs: 4 and 16, respectively;

(e) SEQ ID NOs: 4 and 17, respectively;

(f) SEQ ID NOs: 8 and 14, respectively;

(g) SEQ ID NOs: 7 and 17, respectively;

(h) SEQ ID NOs: 6 and 15, respectively;

(i) SEQ ID NOs: 6 and 5, respectively;

(j) SEQ ID NOs: 7 and 5, respectively;

(k) SEQ ID NOs: 8 and 5, respectively;

(l) SEQ ID NOs: 9 and 5, respectively;

(m) SEQ ID NOs: 10 and 5, respectively;

(n) SEQ ID NOs: 11 and 5, respectively;

(o) SEQ ID NOs: 6 and 14, respectively;

(p) SEQ ID NOs: 6 and 16, respectively;

(q) SEQ ID NOs: 6 and 17, respectively;

(r) SEQ ID NOs: 7 and 14, respectively;

(s) SEQ ID NOs: 7 and 15, respectively;

(t) SEQ ID NOs: 7 and 16, respectively;

(u) SEQ ID NOs: 8 and 15, respectively;

(v) SEQ ID NOs: 8 and 16, respectively;

(w) SEQ ID NOs: 8 and 17, respectively;

(x) SEQ ID NOs: 9 and 14, respectively;

(y) SEQ ID NOs: 9 and 15, respectively;

(z) SEQ ID NOs: 9 and 17, respectively;

(aa) SEQ ID NOs: 10 and 14, respectively;

(bb) SEQ ID NOs: 10 and 15, respectively;

(cc) SEQ ID NOs: 10 and 16, respectively;

(dd) SEQ ID NOs: 10 and 17, respectively;

(ee) SEQ ID NOs: 11 and 14, respectively;

(ff) SEQ ID NOs: 11 and 15, respectively;

(gg) SEQ ID NOs: 11 and 16, respectively; and

(hh) SEQ ID NOs: 11 and 17, respectively.

Some aspects of the disclosure relate to any of the preceding isolatedmonoclonal antibodies, or antigen binding portions thereof, in which theantibody is a humanized antibody.

Other aspects of the disclosure relate to any of the preceding isolatedmonoclonal antibodies, or antigen binding portions thereof, in which theantibody has neutralizing activity against Zika virus.

Some aspects of the disclosure relate to any of the preceding isolatedmonoclonal antibodies, or antigen binding portions thereof, in which theantibody is selected from the group consisting of an IgG1, an IgG2, anIgG3, an IgG4, an IgM, an IgA1, an IgA2, an IgD, and an IgE antibody. Insome aspects of the disclosure, any of the preceding isolated monoclonalantibodies, or antigen binding portions thereof, is an IgG1 antibody.

Other aspects of the disclosure relate to a pharmaceutical compositioncomprising any of the preceding isolated monoclonal antibodies, orantigen binding portions thereof, and a pharmaceutically acceptablecarrier.

Another aspect of the disclosure relates to a method for treating Zikavirus infection in a subject in need thereof, comprising administeringan effective amount of any of the preceding isolated monoclonalantibodies, or antigen binding portions thereof, or a pharmaceuticalcomposition comprising any of the preceding isolated monoclonalantibodies, or antigen binding portions thereof, and a pharmaceuticallyacceptable carrier.

Another aspect of the disclosure relates to a method for preventing Zikavirus infection in a subject, comprising administering an effectiveamount of any of the preceding isolated monoclonal antibodies, orantigen binding portions thereof, or a pharmaceutical compositioncomprising any of the preceding isolated monoclonal antibodies, orantigen binding portions thereof, and a pharmaceutically acceptablecarrier.

In other aspects, the disclosure relates to methods for treating orpreventing vertical infection to a fetus in a pregnant subject,comprising administering an effective amount of any of the precedingisolated monoclonal antibodies, or antigen binding portions thereof, ora pharmaceutical composition comprising any of the preceding isolatedmonoclonal antibodies, or antigen binding portions thereof, and apharmaceutically acceptable carrier. In some aspects, the disclosurerelates to methods for reducing or reducing the risk of verticalinfection to a fetus in a pregnant subject, comprising administering aneffective amount of any of the preceding isolated monoclonal antibodies,or antigen binding portions thereof, or a pharmaceutical compositioncomprising any of the preceding isolated monoclonal antibodies, orantigen binding portions thereof, and a pharmaceutically acceptablecarrier.

In another aspect, the disclosure relates to methods for treating orpreventing fetal Zika virus infection, comprising administering to apregnant subject in need thereof, an effective amount of any of thepreceding isolated monoclonal antibodies, or antigen binding portionsthereof, or a pharmaceutical composition comprising any of the precedingisolated monoclonal antibodies, or antigen binding portions thereof, anda pharmaceutically acceptable carrier. In some aspects, the disclosurerelates to methods for reducing or reducing the risk of fetal Zika virusinfection, comprising administering to a pregnant subject an effectiveamount of any of the preceding isolated monoclonal antibodies, orantigen binding portions thereof, or a pharmaceutical compositioncomprising any of the preceding isolated monoclonal antibodies, orantigen binding portions thereof, and a pharmaceutically acceptablecarrier.

Another aspect of the disclosure relates to methods for treating orpreventing fetal mortality in a pregnant subject, comprisingadministering to the subject an effective amount of any of the precedingisolated monoclonal antibodies, or antigen binding portions thereof, ora pharmaceutical composition comprising any of the preceding isolatedmonoclonal antibodies, or antigen binding portions thereof, and apharmaceutically acceptable carrier. In some aspects, the disclosurerelates to methods for reducing or reducing the risk of fetal mortalityin a pregnant subject, comprising administering an effective amount ofany of the preceding isolated monoclonal antibodies, or antigen bindingportions thereof, or a pharmaceutical composition comprising any of thepreceding isolated monoclonal antibodies, or antigen binding portionsthereof, and a pharmaceutically acceptable carrier.

In other aspects, the disclosure relates to methods for treating orpreventing placental Zika virus infection, comprising administering to apregnant subject in need thereof, an effective amount of any of thepreceding isolated monoclonal antibodies, or antigen binding portionsthereof, or a pharmaceutical composition comprising any of the precedingisolated monoclonal antibodies, or antigen binding portions thereof, anda pharmaceutically acceptable carrier. In some aspects, the disclosurerelates to methods for reducing or reducing the risk of placental Zikavirus infection, comprising administering to a pregnant subject aneffective amount of any of the preceding isolated monoclonal antibodies,or antigen binding portions thereof, or a pharmaceutical compositioncomprising any of the preceding isolated monoclonal antibodies, orantigen binding portions thereof, and a pharmaceutically acceptablecarrier.

In any of the foregoing methods, the pregnant subject is infected withZika virus. In some aspects, the pregnant subject is at risk of beinginfected with Zika virus.

Some aspects of the disclosure relate to a nucleic acid comprising anucleotide sequence encoding the light chain, heavy chain, or both lightand heavy chains of any of the preceding isolated monoclonal antibodies,or antigen binding portions thereof. In some aspects, the disclosurerelates to an expression vector comprising the nucleic acid. In furtheraspects, the disclosure relates to a cell transformed with theexpression vector.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the heavy chain sequence for anti-TDRD3 antibody (top) andthe heavy chain sequences for the anti-Zika antibodies generated(bottom). Modifications are bold and underlined.

FIG. 1B shows the light chain sequence for anti-TDRD3 antibody (top) andthe light chain sequences for the anti-Zika antibodies generated(bottom). Modifications are bold and underlined.

FIG. 2 shows binding data of anti-Zika antibodies to different strainsof Zika virus as measured by a sandwich ELISA.

FIGS. 3A and 3B are graphs showing the percent neutralization byanti-Zika antibodies determined in a plaque reduction neutralizationtest using the Zika virus ILM strain (Brazil Paraiba 2015) (3A) orH/PF/2013 strain (3B).

FIGS. 4A-4D are graphs showing body weight over time (4A and 4B) andsurvival over time (4C and 4D) in mice with Zika virus infection(H/PF/2013 strain) treated either prophylactically (4A and 4C) ortherapeutically (4B and 4D) with anti-Zika antibodies.

FIG. 5 is graphs depicting viral load over time in mice with Zika virusinfection (H/PF/2013 strain) treated either prophylactically (top) ortherapeutically (bottom) with anti-Zika antibodies.

FIG. 6A is a line graph showing the weight, in grams, of mice over timetreated with varying doses of mAb 8 administered a day after Zika virusinfection (H/PF/2013 strain).

FIG. 6B is a line graph depicting viral load over time in mice treatedwith varying doses of mAb 8 administered a day after Zika virusinfection (H/PF/2013 strain).

FIG. 6C is a Kaplan-Meier graph showing survival of mice treated withvarying doses of mAb 8 administered a day after Zika virus infection(H/PF/2013 strain).

FIG. 7A is a graph depicting antibody-dependent enhancement activity ofvarious antibodies in THP-1 cells infected with Zika virus (H/PF/2013strain). Plaque titers resulting from opsonization with differentconcentrations of antibodies is shown.

FIG. 7B provides a comparison of peak enhancement titers (viral titer atpoint of greatest enhancement observed) resulting from the variousantibodies as indicated. *<0.05 (non-parametric, two-tailed student'sT-test).

FIG. 8A is a schematic providing the study design for assessing verticalinfection and fetal mortality in pregnant A129 mice infected with Zikavirus (H/PF/2013 strain).

FIGS. 8B-8F provide graphs showing the results of the study described inFIG. 8A. Specifically, levels of viral RNA in the mothers at day 2 (8B)or day 7 (8C), percent fetus survival as measured by percent lethality(8D), and viral RNA in fetuses (8E) or placenta (8F) from mice treatedwith an isotype control IgG or mAb 8 are shown.

DETAILED DESCRIPTION Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “Zika virus”, refers to members of the family Flaviviridae, andis normally associated with mild symptoms including fever, rash jointpain or conjunctivitis, but has recently been linked to microcephaly innewborn babies by mother-to-child transmission and Guillain-Barresyndrome. The genome of Zika virus consists of a single strand ofpositive sense RNA that is approximately 11 kb in length. Zika virions,like virions of other falviviruses, contain 10 functionally distinctproteins, including three structural proteins incorporated into thevirus particle.

The term “Zika virus envelope protein (“E” or “EP”)” refers to theprotein responsible for viral entry and virion budding. It is composedof three distinct domains. E Domain I (E-DI) is the central domain thatorganizes the entire E protein structure. E Domain II (E-DII) is formedfrom two extended loops projecting from E-DI and lies in a pocket at theE-DI and E Domain III (E-DIII). E-DIII is an immunoglobulin-like domainthat forms small protrusions on the surface of an otherwise smoothspherical mature virus particle, and is thought to interact withcellular receptors on target cells. At the distal end of E-DII is aglycine-rich, hydrophobic sequences referred to as the “fusion loop.”The fusion loop encompasses residues 98 to 110 and is highly conservedamong flaviviruses. In certain embodiments, “Zika virus envelopeprotein” refers to the fusion loop. In certain embodiments, the fusionloop has the amino acid sequence set forth in SEQ ID NO: 3.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers, in certain embodiments, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., Zika virus EP). Such “fragments” are, for example between about 8and about 1500 amino acids in length, suitably between about 8 and about745 amino acids in length, suitably about 8 to about 300, for exampleabout 8 to about 200 amino acids, or about 10 to about 50 or 100 aminoacids in length. It has been shown that the antigen-binding function ofan antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), CL and CH1 domains;(ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. Antigen-bindingportions can be produced by recombinant DNA techniques, or by enzymaticor chemical cleavage of intact immunoglobulins.

The term “monoclonal antibody,” as used herein, refers to an antibodywhich displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody which displays a single binding specificity andwhich has variable and optional constant regions derived from humangermline immunoglobulin sequences. In one embodiment, human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizeparticular human germline immunoglobulin sequences are encoded by thegermline genes, but include subsequent rearrangements and mutationswhich occur, for example, during antibody maturation. As known in theart (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for a foreignantigen. In addition to rearrangement, the variable region can befurther modified by multiple single amino acid changes (referred to assomatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid molecules that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not have sequence identity with the original nucleicacid molecules, but instead will be substantially identical or similar(i.e., have at least 80% identity).

The term “human antibody” includes antibodies having variable andconstant regions (if present) of human germline immunoglobulinsequences. Human antibodies of the disclosure can include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo) (see, Lonberg, N. et al. (1994) Nature368(6474): 856-859); Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y. Acad. Sci 764:536-546). However, the term “human antibody” does notinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences (i.e., humanized antibodies).

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. This termrefers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

As used herein, “neutralizing antibody” refers to an antibody, forexample, a monoclonal antibody, capable of disrupting a formed viralparticle or inhibiting formatting of a viral particle or prevention ofbinding to or infection of mammalian cells by a viral particle.

In some embodiments, the antibodies described herein neutralize Zikavirus.

As used herein, “diagnostic antibody” or “detection antibody” or“detecting antibody” refers to an antibody, for example, a monoclonalantibody, capable of detecting the presence of an antigenic targetwithin a sample. As will be appreciated by one of skill in the art, suchdiagnostic antibodies preferably have high specificity for theirantigenic target.

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or“substantially human” means that, when aligned to a human immunoglobulinor antibody amino acid sequence for comparison purposes, the regionshares at least 80-90%, preferably at least 90-95%, more preferably atleast 95-99% identity (i.e., local sequence identity) with the humanframework or constant region sequence, allowing, for example, forconservative substitutions, consensus sequence substitutions, germlinesubstitutions, back-mutations, and the like. The introduction ofconservative substitutions, consensus sequence substitutions, germlinesubstitutions, back-mutations, and the like, is often referred to as“optimization” of a humanized antibody or chain. The phrase“substantially from a non-human immunoglobulin or antibody” or“substantially non-human” means having an immunoglobulin or antibodysequence at least 80-95%, preferably at least 90-95%, more preferably,96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., anon-human mammal.

Referring to the well-recognized nomenclature for amino acids, the threeletter code, or one letter code, is used, including the codes “Xaa” or“X” to indicate any amino acid residue. Thus, Xaa or X may typicallyrepresent any of the 20 naturally occurring amino acids.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For purposes of classifying aminoacids substitutions as conservative or nonconservative, amino acids aregrouped as follows: Group I (hydrophobic sidechains): leu, met, ala,val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser,thr; Group III (acidic side chains): asp, glu; Group IV (basic sidechains): asn, gin, his, lys, arg; Group V (residues influencing chainorientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

A mutation (e.g., a back-mutation) is said to substantially affect theability of a heavy or light chain to direct antigen binding if itaffects (e.g., decreases) the binding affinity of an intactimmunoglobulin or antibody (or antigen binding fragment thereof)comprising said chain by at least an order of magnitude compared to thatof the antibody (or antigen binding fragment thereof) comprising anequivalent chain lacking said mutation. A mutation “does notsubstantially affect (e.g., decrease) the ability of a chain to directantigen binding” if it affects (e.g., decreases) the binding affinity ofan intact immunoglobulin or antibody (or antigen binding fragmentthereof) comprising said chain by only a factor of two, three, or fourof that of the antibody (or antigen binding fragment thereof) comprisingan equivalent chain lacking said mutation.

In certain embodiments, humanized immunoglobulins or antibodies bindantigen with an affinity that is within a factor of three, four, or fiveof that of the corresponding non-humanized antibody. For example, if thenonhumanized antibody has a binding affinity of 10⁹ M⁻¹, humanizedantibodies will have a binding affinity of at least 3 times 10⁹ M⁻¹, 4times 10⁹ M⁻¹ or 10⁹ M⁻¹. When describing the binding properties of animmunoglobulin or antibody chain, the chain can be described based onits ability to “direct antigen (e.g., Zika virus EP) binding”. A chainis said to “direct antigen binding” when it confers upon an intactimmunoglobulin or antibody (or antigen binding fragment thereof) aspecific binding property or binding affinity.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species. The terms “humanized immunoglobulin” or “humanizedantibody” are not intended to encompass chimeric immunoglobulins orantibodies, as defined infra. Although humanized immunoglobulins orantibodies are chimeric in their construction (i.e., comprise regionsfrom more than one species of protein), they include additional features(i.e., variable regions comprising donor CDR residues and acceptorframework residues) not found in chimeric immunoglobulins or antibodies,as defined herein.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to Zika virus EP is substantially free of antibodiesthat specifically bind antigens other than Zika virus EP). An isolatedantibody is typically substantially free of other cellular materialand/or chemicals. In certain embodiments of the disclosure, acombination of“isolated” antibodies having different Zika virus EPspecificities is combined in a well-defined composition.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids in a unique spatial conformation. Methods for determiningwhat epitopes are bound by a given antibody (i.e., epitope mapping) arewell known in the art and include, for example, immunoblotting andimmunoprecipitation assays, wherein overlapping or contiguous peptidesfrom Zika virus EP are tested for reactivity with the given anti-EPantibody. Methods of determining spatial conformation of epitopesinclude techniques in the art and those described herein, for example,x-ray crystallography and 2-dimensional nuclear magnetic resonance (see,e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, G. E. Morris, Ed. (1996)).

Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen, i.e., a competitive binding assay.Competitive binding is determined in an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen. Numerous types of competitive bindingassays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using 1-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

The term “epitope mapping” refers to the process of identification ofthe molecular determinants for antibody-antigen recognition. Numerousmethods for epitope mapping are known in the art, such as x-rayanalysis, protease mapping, hydrogen/deuterium exchange massspectrometry (HDX-MS), 2D nuclear magnetic resonance, alanine scanning,and deep mutational scanning.

To facilitate engineering of antibodies that target the Zika virusenvelope protein (EP), epitope hotspots were determined by analyzing thepercent buried surface area of interface residues. In some embodiments,the anti-Zika virus antibodies described herein bind an epitope on thefusion loop comprising residues D98, R99 and W101 (SEQ ID NO: 3).

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). For example, the Kd can be about 200 nM, 150nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM,2 nM, 1 nM, or stronger. Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present disclosure.

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen. Typically, the antibody bindswith an equilibrium dissociation constant (K_(D)) of approximately lessthan 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or 10^(t10)M or even lower when determined by surface plasmon resonance (SPR)technology in a BIACORE 2000 instrument using recombinant Zika virus EPas the analyte and the antibody as the ligand and binds to thepredetermined antigen with an affinity that is at least two-fold greaterthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. The phrases “an antibody recognizing an antigen” and “anantibody specific for an antigen” are used interchangeably herein withthe term “an antibody which binds specifically to an antigen.”

The term “K_(D),” as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

The term “kd” as used herein, is intended to refer to the off rateconstant for the dissociation of an antibody from the antibody/antigencomplex.

The term “ka” as used herein, is intended to refer to the on rateconstant for the association of an antibody with the antigen.

The term “EC50,” as used herein, refers to the concentration of anantibody or an antigen-binding portion thereof, which induces aresponse, either in an in vitro or an in vivo assay, which is 50% of themaximal response, i.e., halfway between the maximal response and thebaseline.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. In oneembodiment, a human monoclonal antibody of the disclosure is of the IgG1isotype. In certain embodiments, the human IgG1 has a heavy chainconstant domain sequence as set forth in SEQ ID NO: 1 and a light chainconstant domain sequence as set forth in SEQ ID NO: 2.

The term “binds to Zika virus EP,” refers to the ability of an antibodydescribed herein to bind to Zika virus EP, for example, expressed on thesurface of a cell or which is attached to a solid support.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The present disclosure also encompasses “conservative sequencemodifications” of the sequences set forth in SEQ ID NOs: 4-53 i.e.,amino acid sequence modifications which do not abrogate the binding ofthe antibody encoded by the nucleotide sequence or containing the aminoacid sequence, to the antigen. Such conservative sequence modificationsinclude conservative nucleotide and amino acid substitutions, as wellas, nucleotide and amino acid additions and deletions. For example,modifications can be introduced into SEQ ID NOs: 4-53 by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions includeones in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, a predicted nonessential amino acidresidue in an anti-EP antibody is preferably replaced with another aminoacid residue from the same side chain family. Methods of identifyingnucleotide and amino acid conservative substitutions which do noteliminate antigen binding are well-known in the art (see, e.g., Brummellet al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng.12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA94:412-417 (1997)).

Alternatively, in certain embodiments, mutations can be introducedrandomly along all or part of an anti-EP antibody coding sequence, suchas by saturation mutagenesis, and the resulting modified anti-EPantibodies can be screened for binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions x 100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present disclosure canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the disclosure. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to the protein molecules of thedisclosure. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

When given an amino acid sequence, one versed in the art can makeconservative substitutions to the nucleotide sequence encoding itwithout altering the amino acid sequence, given the redundancy in thegenetic code. The nucleic acid compositions, while often in a nativesequence (except for modified restriction sites and the like), fromeither cDNA, genomic or mixtures thereof may be mutated, in accordancewith standard techniques to provide gene sequences. For codingsequences, these mutations, may affect amino acid sequence as desired.In particular, DNA sequences substantially homologous to or derived fromnative V, D, J, constant, switches and other such sequences describedherein are contemplated (where “derived” indicates that a sequence isidentical or modified from another sequence).

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the disclosure is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, in need of suchtreatment, a human antibody of the present disclosure, for example, asubject in need of an enhanced immune response against a particularantigen (e.g., Zika virus) or a subject who ultimately may acquire sucha disorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disorder or recurring disorder,or in order to prolong the survival of a subject beyond that expected inthe absence of such treatment.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the disorderbeing treated and the general state of the patient's own immune system.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentdisclosure can be used to treat a subject with an immune disorder. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc. In some embodiments, the subject is pregnant.

As used herein, the term “vertical infection” refers to mother-to-childtransmission of a pathogen (e.g., Zika virus). In some embodiments, theanti-Zika virus EP antibodies described herein prevent verticalinfection in a pregnant subject.

Various aspects of the disclosure are described in further detail in thefollowing subsections.

Production of Antibodies to Zika Virus Envelope Protein

The present disclosure encompasses antibodies that bind Zika virus EP.In some embodiments, antibodies that bind Zika virus EP are optimizedmonoclonal antibodies which include CDRs, or optimized CDRs, based on ananti-TDRD3 (Tudor Domain Containing 3) human monoclonal antibody.Provided herein are isolated monoclonal antibodies or antigen bindingportions thereof, comprising heavy and light chain variable regionsequences comprising (further described in Tables 2 and 3):

(a) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 20, 26 and 31, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,respectively;

(b) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 22, 28 and 33, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,respectively;

(c) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 20, 26 and 31, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,respectively;

(d) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 20, 26 and 31, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,respectively;

(e) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 28 and 32, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,respectively;

(f) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 27 and 32, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,respectively;

(g) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 27 and 32, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,respectively;

(h) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 27 and 32, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,respectively;

(i) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 28 and 32, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,respectively;

(j) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 22, 28 and 33, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,respectively;

(k) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 23, 28 and 34, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,respectively;

(l) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 28 and 34, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,respectively;

(m) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 27 and 32, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,respectively;

(n) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 28 and 32, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,respectively;

(o) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 28 and 32, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,respectively;

(p) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 22, 28 and 33, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,respectively;

(q) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 22, 28 and 33, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,respectively;

(r) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 23, 28 and 34, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,respectively;

(s) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 23, 28 and 34, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,respectively;

(t) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 23, 28 and 34, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,respectively;

(u) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 28 and 34, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,respectively;

(v) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 28 and 34, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,respectively; and

(w) a heavy chain comprising CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 21, 28 and 34, respectively, and a light chain comprisingCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,respectively.

In some embodiments, antibodies that bind Zika virus EP are optimizedmonoclonal antibodies which include heavy and/or light chain variableregions, or optimized heavy and/or light chain variable regions, basedon an anti-TDRD3 human monoclonal antibody.

Also provided herein, are isolated monoclonal antibodies or antigenbinding portions thereof, comprising heavy and light chain variablesequences comprising:

(a) SEQ ID NOs: 4 and 14, respectively;

(b) SEQ ID NOs: 4 and 15, respectively;

(c) SEQ ID NOs: 9 and 16, respectively;

(d) SEQ ID NOs: 4 and 16, respectively;

(e) SEQ ID NOs: 4 and 17, respectively;

(f) SEQ ID NOs: 8 and 14, respectively;

(g) SEQ ID NOs: 7 and 17, respectively;

(h) SEQ ID NOs: 6 and 15, respectively;

(i) SEQ ID NOs: 6 and 5, respectively;

(j) SEQ ID NOs: 7 and 5, respectively;

(k) SEQ ID NOs: 8 and 5, respectively;

(l) SEQ ID NOs: 9 and 5, respectively;

(m) SEQ ID NOs: 10 and 5, respectively;

(n) SEQ ID NOs: 11 and 5, respectively;

(o) SEQ ID NOs: 6 and 14, respectively;

(p) SEQ ID NOs: 6 and 16, respectively;

(q) SEQ ID NOs: 6 and 17, respectively;

(r) SEQ ID NOs: 7 and 14, respectively;

(s) SEQ ID NOs: 7 and 15, respectively;

(t) SEQ ID NOs: 7 and 16, respectively;

(u) SEQ ID NOs: 8 and 15, respectively;

(v) SEQ ID NOs: 8 and 16, respectively;

(w) SEQ ID NOs: 8 and 17, respectively;

(x) SEQ ID NOs: 9 and 14, respectively;

(y) SEQ ID NOs: 9 and 15, respectively;

(z) SEQ ID NOs: 9 and 17, respectively; (aa) SEQ ID NOs: 10 and 14,respectively; (bb) SEQ ID NOs: 10 and 15, respectively; (cc) SEQ ID NOs:10 and 16, respectively; (dd) SEQ ID NOs: 10 and 17, respectively; (ee)SEQ ID NOs: 11 and 14, respectively; (if) SEQ ID NOs: 11 and 15,respectively; (gg) SEQ ID NOs: 11 and 16, respectively; and (hh) SEQ IDNOs: 11 and 17, respectively.

Monoclonal antibodies described herein can be produced using a varietyof known techniques, such as the standard somatic cell hybridizationtechnique described by Kohler and Milstein, Nature 256: 495 (1975).Although somatic cell hybridization procedures are preferred, inprinciple, other techniques for producing monoclonal antibodies also canbe employed, e.g., viral or oncogenic transformation of B lymphocytes,phage display technique using libraries of human antibody genes.

Accordingly, in certain embodiments, a hybridoma method is used forproducing an antibody that binds Zika virus EP. In this method, a mouseor other appropriate host animal can be immunized with a suitableantigen in order to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the antigen used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes can then be fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Culture medium in which hybridoma cells are growing isassayed for production of monoclonal antibodies directed against theantigen. After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones can be separated fromthe culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

In certain embodiments, antibodies and antibody portions that bind Zikavirus EP can be isolated from antibody phage libraries generated usingthe techniques described in, for example, McCafferty et al., Nature,348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991), Markset al., J. Mol. Biol., 222:581-597 (1991) and Hoet et al (2005) NatureBiotechnology 23, 344-348; U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S.Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al. Additionally,production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21:2265-2266 (1993)) may also be used.

In certain embodiments, the antibody that binds Zika virus EP isproduced using the phage display technique described by Hoet et al.,supra. This technique involves the generation of a human Fab libraryhaving a unique combination of immunoglobulin sequences isolated fromhuman donors and having synthetic diversity in the heavy-chain CDRs isgenerated. The library is then screened for Fabs that bind to Zika virusEP.

The preferred animal system for generating hybridomas which produceantibodies of the disclosure is the murine system. Hybridoma productionin the mouse is well known in the art, including immunization protocolsand techniques for isolating and fusing immunized splenocytes.

In certain embodiments, antibodies directed against Zika virus EP aregenerated using transgenic or transchromosomal mice carrying parts ofthe human immune system rather than the mouse system. In someembodiments, antibodies described herein are generated using transgenicmice, referred to herein as “HuMAb mice” which contain a humanimmunoglobulin gene miniloci that encodes unrearranged human heavy (μand γ) and κ light chain immunoglobulin sequences, together withtargeted mutations that inactivate the endogenous μ and κ chain loci(Lonberg, N. et al. (1994) Nature 368(6474): 856-859). Accordingly, themice exhibit reduced expression of mouse IgM or κ, and in response toimmunization, the introduced human heavy and light chain transgenesundergo class switching and somatic mutation to generate high affinityhuman IgGκ monoclonal antibodies (Lonberg, N. et al. (1994), supra;reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol.13: 65-93, and Harding, F. and Lonberg, N. (1995)Ann. N.Y. Acad. Sci764:536-546). The preparation of HuMAb mice is described in detail belowand in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295;Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon etal. (1993) Proc. Natl. Acad. Sci USA 90:3720-3724; Choi et al. (1993)Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830;Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg et al., (1994)Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Taylor, L. et al. (1994) InternationalImmunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y.Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology14: 845-851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; and 5,770,429; all to Lonberg and Kay, and GenPharmInternational; U.S. Pat. No. 5,545,807 to Surani et al.; InternationalPublication Nos. WO 98/24884, published on Jun. 11, 1998; WO 94/25585,published Nov. 10, 1994; WO 93/1227, published Jun. 24, 1993; WO92/22645, published Dec. 23, 1992; WO 92/03918, published Mar. 19,1992).

In certain embodiments, antibodies described herein can be raised usinga mouse that carries human immunoglobulin sequences on transgenes andtranschomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto in the art as “KM mice”, are described in detail in PCT PublicationWO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-Zika virus EP antibodies of the disclosure. For example, analternative transgenic system referred to as the Xenomouse (Abgenix,Inc.) can be used; such mice are described in, for example, U.S. Pat.Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 toKucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-Zika virus EP antibodies described herein. For example, micecarrying both a human heavy chain transchromosome and a human lightchain tranchromosome, referred to in the art as “TC mice” can be used;such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci.USA 97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-Zikavirus EP antibodies of the disclosure.

Additional mouse systems described in the art for raising humanantibodies also can be applied to raising anti-Zika virus EP antibodiesof the disclosure, including but not limited to (i) the VelocImmune®mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouseheavy and light chain variable regions have been replaced, viahomologous recombination, with human heavy and light chain variableregions, operatively linked to the endogenous mouse constant regions,such that chimeric antibodies (human V/mouse C) are raised in the mice,and then subsequently converted to fully human antibodies using standardrecombinant DNA techniques; and (ii) the MeMo® mouse (MerusBiopharmaceuticals, Inc.), in which the mouse contains unrearrangedhuman heavy chain variable regions but a single rearranged human commonlight chain variable region. Such mice, and use thereof to raiseantibodies, are described in, for example, WO 2009/15777, US2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO2011/163314, WO 2012/148873, US 2012/0070861 and US 2012/0073004.

Human monoclonal antibodies described herein can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

In certain embodiments, the mAbs described herein can be produced inplants using deconstructed viral vectors, as described in Olinger etal., PNAS 2012; 109, 18030-18035, herein incorporated by reference. Incertain embodiments, the mAbs are produced in tobacco plants.

In certain embodiments, chimeric antibodies can be prepared based on thesequence of a murine monoclonal antibodies described herein. A chimericantibody refers to an antibody whose light and heavy chain genes havebeen constructed, typically by genetic engineering, from immunoglobulingene segments belonging to different species. For example, the variable(V) segments of the genes from a mouse monoclonal antibody may be joinedto human constant (C) segments, such as IgG1 and IgG4. Human isotypeIgG1 is preferred. A typical chimeric antibody is thus a hybrid proteinconsisting of the V or antigen-binding domain from a mouse antibody andthe C or effector domain from a human antibody.

Production of Humanized Antibodies

The term “humanized antibody” refers to an antibody comprising at leastone chain comprising variable region framework residues substantiallyfrom a human antibody chain (referred to as the acceptor immunoglobulinor antibody) and at least one complementarity determining regionsubstantially from a mouse antibody, (referred to as the donorimmunoglobulin or antibody). See, Queen et al., Proc. Natl. Acad. Sci.USA 86:10029-10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.5,585,089, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,693,762, Selick etal., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539 (incorporated byreference in their entirety for all purposes). The constant region(s),if present, are also substantially or entirely from a humanimmunoglobulin.

The substitution of mouse CDRs into a human variable domain framework ismost likely to result in retention of their correct spatial orientationif the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Kettleborough et al., Protein Engineering4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) andCarter et al., WO 92/22653.

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting ahuman-anti-mouse-antibody (HAMA) response in humans. Art-recognizedmethods of determining an immune response can be performed to monitor aHAMA response in a particular patient or during clinical trials.Patients administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution is determined, inpart, by computer modeling. Computer hardware and software are describedherein for producing three-dimensional images of immunoglobulinmolecules. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 50% sequence identity are selectedfor modeling, and preferably those sharing at least 60%, 70%, 80%, 90%sequence identity or more are selected for modeling. The solved startingstructures are modified to allow for differences between the actualamino acids in the immunoglobulin chains or domains being modeled, andthose in the starting structure. The modified structures are thenassembled into a composite immunoglobulin. Finally, the model is refinedby energy minimization and by verifying that all atoms are withinappropriate distances from one another and that bond lengths and anglesare within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g., is within about        3-6 angstroms of a CDR region as determined by computer        modeling), or    -   (4) participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like.

CDR and framework regions are as defined by Kabat et al. or Chothia etal., supra. When framework residues, as defined by Kabat et al., supra,constitute structural loop residues as defined by Chothia et al., supra,the amino acids present in the mouse antibody may be selected forsubstitution into the humanized antibody. Residues which are “adjacentto a CDR region” include amino acid residues in positions immediatelyadjacent to one or more of the CDRs in the primary sequence of thehumanized immunoglobulin chain, for example, in positions immediatelyadjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia(See e.g., Chothia and Lesk J M B 196:901 (1987)). These amino acids areparticularly likely to interact with the amino acids in the CDRs and, ifchosen from the acceptor, to distort the donor CDRs and reduce affinity.Moreover, the adjacent amino acids may interact directly with theantigen (Amit et al., Science, 233:747 (1986), which is incorporatedherein by reference) and selecting these amino acids from the donor maybe desirable to keep all the antigen contacts that provide affinity inthe original antibody.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to affect a CDR region. In certain embodiments,residues that “otherwise interact with a CDR region” are identified byanalyzing a three-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor antibody, shows that certain amino acids outside of theCDRs are close to the CDRs and have a good probability of interactingwith amino acids in the CDRs by hydrogen bonding, Van der Waals forces,hydrophobic interactions, etc. At those amino acid positions, the donorimmunoglobulin amino acid rather than the acceptor immunoglobulin aminoacid may be selected. Amino acids according to this criterion willgenerally have a side chain atom within about 3 angstrom units (A) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above.

In the case of atoms that may form a hydrogen bond, the 3 Angstroms ismeasured between their nuclei, but for atoms that do not form a bond,the 3 Angstroms is measured between their Van der Waals surfaces. Hence,in the latter case, the nuclei must be within about 6 Angstroms (3Angstroms plus the sum of the Van der Waals radii) for the atoms to beconsidered capable of interacting. In many cases the nuclei will be from4 or 5 to 6 Angstroms apart. In determining whether an amino acid caninteract with the CDRs, it is preferred not to consider the last 8 aminoacids of heavy chain CDR 2 as part of the CDRs, because from theviewpoint of structure, these 8 amino acids behave more as part of theframework.

Amino acids that are capable of interacting with amino acids in theCDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact antibody, and (2) in a hypothetical molecule consisting ofthe antibody with its CDRs removed. A significant difference betweenthese numbers of about 10 square angstroms or more shows that access ofthe framework amino acid to solvent is at least partly blocked by theCDRs, and therefore that the amino acid is making contact with the CDRs.Solvent accessible surface area of an amino acid may be calculated basedon a three-dimensional model of an antibody, using algorithms known inthe art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee andRichards, J. Mol. Biol. 55:379 (1971), both of which are incorporatedherein by reference). Framework amino acids may also occasionallyinteract with the CDRs indirectly, by affecting the conformation ofanother framework amino acid that in turn contacts the CDRs.

The amino acids at several positions in the framework are known to becapable of interacting with the CDRs in many antibodies (Chothia andLesk, supra, Chothia et al., supra and Tramontano et al., J. Mol. Biol.215:175 (1990), all of which are incorporated herein by reference).Notably, the amino acids at positions 2, 48, 64 and 71 of the lightchain and 26-30, 71 and 94 of the heavy chain (numbering according toKabat) are known to be capable of interacting with the CDRs in manyantibodies. The amino acids at positions 35 in the light chain and 93and 103 in the heavy chain are also likely to interact with the CDRs. Atall these numbered positions, choice of the donor amino acid rather thanthe acceptor amino acid (when they differ) to be in the humanizedimmunoglobulin is preferred. On the other hand, certain residues capableof interacting with the CDR region, such as the first 5 amino acids ofthe light chain, may sometimes be chosen from the acceptorimmunoglobulin without loss of affinity in the humanized immunoglobulin.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriais substituted. In some embodiments, all or most of the amino acidsfulfilling the above criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. Although not usually desirable, it is sometimespossible to make one or more conservative amino acid substitutions ofCDR residues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin. By conservative substitutions isintended combinations such as gly, ala; val, ile, leu; asp, glu; asn,gln; ser, thr; lys, arg; and phe, tyr.

Additional candidates for substitution are acceptor human frameworkamino acids that are unusual or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. For example,substitution may be desirable when the amino acid in a human frameworkregion of the acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is common for thatposition in human immunoglobulin sequences; or when the amino acid inthe acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. These criteria help ensure that anatypical amino acid in the human framework does not disrupt the antibodystructure. Moreover, by replacing an unusual human acceptor amino acidwith an amino acid from the donor antibody that happens to be typicalfor human antibodies, the humanized antibody may be made lessimmunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20% but usually less than about 10% ofsequences in a representative sample of sequences, and the term“common”, as used herein, indicates an amino acid occurring in more thanabout 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al., supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence.

Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under thealternative definition proposed by Chothia et al., supra. Additionalcandidates for substitution are acceptor human framework amino acidsthat would be identified as part of a CDR region under the AbM and/orcontact definitions.

Additional candidates for substitution are acceptor framework residuesthat correspond to a rare or unusual donor framework residue. Rare orunusual donor framework residues are those that are rare or unusual (asdefined herein) for murine antibodies at that position. For murineantibodies, the subgroup can be determined according to Kabat andresidue positions identified which differ from the consensus. Thesedonor specific differences may point to somatic mutations in the murinesequence which enhance activity. Unusual residues that are predicted toaffect binding are retained, whereas residues predicted to beunimportant for binding can be substituted.

Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorantibody chain (i.e., a human antibody chain sharing significantsequence identity with the donor antibody chain) is aligned to agermline antibody chain (likewise sharing significant sequence identitywith the donor chain), residues not matching between acceptor chainframework and the germline chain framework can be substituted withcorresponding residues from the germline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized immunoglobulins are usually substantiallyidentical, and more usually, identical to the framework regions of thehuman antibodies from which they were derived. Of course, many of theamino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, in one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence identity to a human variable framework regionsequence or consensus of such sequences. In another embodiment, thevariable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequenceidentity to a human variable framework region sequence or consensus ofsuch sequences. In general, however, such substitutions are undesirable.

The humanized antibodies preferably exhibit a specific binding affinityfor antigen of at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually the upperlimit of binding affinity of the humanized antibodies for antigen iswithin a factor of three, four or five of that of the donorimmunoglobulin. Often the lower limit of binding affinity is also withina factor of three, four or five of that of donor immunoglobulin.Alternatively, the binding affinity can be compared to that of ahumanized antibody having no substitutions (e.g., an antibody havingdonor CDRs and acceptor FRs, but no FR substitutions). In suchinstances, the binding of the optimized antibody (with substitutions) ispreferably at least two- to three-fold greater, or three- to four-foldgreater, than that of the unsubstituted antibody. For makingcomparisons, activity of the various antibodies can be determined, forexample, by BIACORE (i.e., surface plasmon resonance using unlabeledreagents) or competitive binding assays.

Generation of Antibodies Having Modified Sequences

In certain embodiments, provided herein are antibodies comprising heavyand light chain variable region CDRs from an anti-TDRD3 monoclonalantibody (SEQ ID NOs: 4 and 5, respectively), wherein at least one CDRcomprises an amino acid substitution, such that the antibody binds toZika virus EP. In certain embodiments, the heavy chain variable regionCDR has an amino acid substitution at position 28, 29, 31, 32, 52, 52A,53, 54, 55, 100, 100A, 100B, 100C, 100D, 100E, 100F, 100G, 100H, 102, orcombination thereof, numbering according to Chothia. In certainembodiments, the amino acid substitution at position 28 is serine. Incertain embodiments, the amino acid substitution at position 28 isthreonine. In certain embodiments, the amino acid at position 28 isdeleted. In certain embodiments, the amino acid substitution at position29 is phenylalanine. In certain embodiments, the amino acid substitutionat position 31 is threonine. In certain embodiments, the amino acidsubstitution at position 32 is tyrosine. In certain embodiments, theamino acid substitution at position 52 is threonine. In certainembodiments, the amino acid substitution at position 52A is glycine. Incertain embodiments, the amino acid substitution at position 53 isglutamic acid. In certain embodiments, the amino acid substitution atposition 54 is glycine. In certain embodiments, the amino acidsubstitution at position 55 is aspartic acid. In certain embodiments,the amino acid at position 99 is deleted. In certain embodiments, theamino acid substitution at position 100 is serine tyrosine. In certainembodiments, the amino acid substitution at position 100A is serine. Incertain embodiments, the amino acid substitution at position 100A isthreonine. In certain embodiments, the amino acid substitution atposition 100B is asparagine. In certain embodiments, the amino acidsubstitution at position 100C is phenylalanine. In certain embodiments,the amino acid substitution at position 100D is tyrosine. In certainembodiments, the amino acid substitution at position 100E is tyrosine.In certain embodiments, the amino acid substitution at position 100F istyrosine. In certain embodiments, the amino acid substitution atposition 100G is tyrosine. In certain embodiments, the amino acidsubstitution at position 100H is threonine. In certain embodiments, theamino acid substitution at position 102 is alanine. In certainembodiments, the amino acid substitution at position 102 is valine.

In certain embodiments, the light chain variable region CDR has an aminoacid substitution at positions 26, 29, 31, 32, 33, 50, 53, 54, 55, 56,91, 93, 94, 95, 95B, 96, 97, or combinations thereof, numberingaccording to Chothia. In certain embodiments, the amino acidsubstitution at position 26 is threonine. In certain embodiments, theamino acid substitution at position 29 is isoleucine. In certainembodiments, the amino acid substitution at position 31 is valine. Incertain embodiments, the amino acid substitution at position 32 isphenylalanine. In certain embodiments, the amino acid substitution atposition 33 is leucine. In certain embodiments, the amino acidsubstitution at position 50 is aspartic acid. In certain embodiments,the amino acid substitution at position 53 is threonine. In certainembodiments, the amino acid substitution at position 54 is arginine. Incertain embodiments, the amino acid substitution at position 54 isasparagine. In certain embodiments, the amino acid substitution atposition 55 is alanine. In certain embodiments, the amino acidsubstitution at position 56 is threonine. In certain embodiments, theamino acid substitution at position 91 is arginine. In certainembodiments, the amino acid substitution at position 93 is tyrosine. Incertain embodiments, the amino acid substitution at position 94 isasparagine. In certain embodiments, the amino acid substitution atposition 95 is tryptophan. In certain embodiments, the amino acidsubstitution at position 95B is proline. In certain embodiments, theamino acid substitution at position 96 is tyrosine. In certainembodiments, the amino acid substitution at position 97 is serine. Incertain embodiments, a proline is added between amino acids at positions95B and 96.

In certain embodiments, the antibodies described herein comprise heavyand light chain variable region CDRs, wherein heavy chain variableregion CDR1 has the amino acid sequence GFX₁FSTY (SEQ ID NO: 54),wherein X₁ may or may not be present and is selected from serine andthreonine; wherein heavy chain variable region CDR2 has the amino acidsequence X₂GEGDS (SEQ ID NO: 55), wherein X₂ is selected from serine andthreonine; and wherein heavy chain variable region CDR3 has the aminoacid sequence GYX₃NFYYYTMDX₄ (SEQ ID NO: 56), wherein X₃ is selectedfrom serine and threonine, and X₄ is selected from alanine and valine.

In certain embodiments, heavy chain CDR1 has the amino acid sequenceGFSFSTY (SEQ ID NO: 21). In certain embodiments, heavy chain CDR1 hasthe amino acid sequence GFTGSTY (SEQ ID NO: 22). In certain embodiments,heavy chain CDR1 has the amino acid sequence GFFSTY (SEQ ID NO: 23). Incertain embodiments, heavy chain CDR2 has the amino acid sequence TGEGDS(SEQ ID NO: 28). In certain embodiments, heavy chain CDR2 has the aminoacid sequence SGEGDS (SEQ ID NO: 27). In certain embodiments, heavychain CDR3 has the amino acid sequence GYSNFYYYYTMDA (SEQ ID NO: 32). Incertain embodiments, heavy chain CDR3 has the amino acid sequenceGYSNFYYYYTMDV (SEQ ID NO: 33). In certain embodiments, heavy chain CDR3has the amino acid sequence GYTNFYYYTMDA (SEQ ID NO: 34).

In certain embodiments, the antibodies described herein comprise heavyand light chain variable region CDRs, wherein light chain variableregion CDR1 has the amino acid sequence RAX₅QSIX₆TFLA (SEQ ID NO: 57),wherein X₅ is selected from serine and threonine, and wherein X₆ isselected from serine and valine; wherein light chain variable regionCDR2 has the amino acid sequence DASTX₇AX₅ (SEQ ID NO: 56), wherein X₇is selected from arginine and asparagine, and X₈ is selected from serineand threonine; and wherein light chain variable region CDR3 has theamino acid sequence QQRYNWPPYX₉ (SEQ ID NO: 56), wherein X₉ is selectedfrom serine and threonine.

In certain embodiments, light chain CDR1 has the amino acid sequenceRATQSISTFLA (SEQ ID NO: 38). In certain embodiments, light chain CDR1has the amino acid sequence RASQSISTFLA (SEQ ID NO: 39). In certainembodiments, light chain CDR1 has the amino acid sequence RATQSIVTFLA(SEQ ID NO: 40). In certain embodiments, light chain CDR2 has the aminoacid sequence DASTRAS (SEQ ID NO: 44). In certain embodiments, lightchain CDR2 has the amino acid sequence DASTRAT (SEQ ID NO: 45). Incertain embodiments, light chain CDR2 has the amino acid sequenceDASTNAS (SEQ ID NO: 46). In certain embodiments, light chain CDR3 hasthe amino acid sequence QQRYNWPPYS (SEQ ID NO: 50). In certainembodiments, light chain CDR3 has the amino acid sequence QQRYNWPPYT(SEQ ID NO: 51).

In certain embodiments, the anti-Zika virus EP antibodies describedherein contain framework mutations in the variable region sequences. Insome embodiments, the antibodies described herein comprise heavy andlight chain variable region sequences, wherein the heavy chain variableregion sequence comprises amino acid substitutions at positions 1, 5,23, 33, 38, 47, 49, 50, 57, 58, 68, 71, 73, 78, 80, 81, 82B, 82C, 84,93, 108, or combinations thereof, numbering according to Chothia. Incertain embodiments, the amino acid substitution at position 1 isglutamine. In some embodiments, the amino acid substitution at position5 is leucine. In some embodiments, the amino acid substitution atposition 23 is serine. In some embodiments, the amino acid substitutionat position 33 is serine. In some embodiments, the amino acidsubstitution at position 38 is lysine. In some embodiments, the aminoacid substitution at position 47 is tyrosine. In some embodiments, theamino acid substitution at position 49 is serine. In some embodiments,the amino acid substitution at position 50 is alanine. In someembodiments, the amino acid substitution at position 57 is alanine. Insome embodiments, the amino acid substitution at position 58 isphenylalanine. In some embodiments, the amino acid substitution atposition 68 is glutamic acid. In some embodiments, the amino acidsubstitution at position 71 is arginine. In some embodiments, the aminoacid substitution at position 73 is asparagine. In some embodiments, theamino acid substitution at position 78 is leucine. In some embodiments,the amino acid substitution at position 80 is phenylalanine. In someembodiments, the amino acid substitution at position 81 is glutamicacid. In some embodiments, the amino acid substitution at position 82(B)is lysine. In some embodiments, the amino acid substitution at position82(C) is valine. In some embodiments, the amino acid substitution atposition 84 is proline. In some embodiments, the amino acid substitutionat position 93 is valine. In some embodiments, the amino acidsubstitution at position 108 is serine. In some embodiments, the aminoacid substitution at position 108 is threonine. In some embodiments, theamino acid substitution at position 108 is methionine.

In certain embodiments, the anti-Zika virus EP antibodies describedherein contain framework mutations in the variable region sequences. Insome embodiments, the antibodies described herein comprise heavy andlight chain variable region sequences, wherein the light chain variableregion sequence comprises amino acid substitutions at positions 1, 3, 4,9, 10, 13, 15, 17, 19, 21, 22, 38, 42, 45, 58, 60, 76, 77, 79, 85, 104,or combinations thereof, numbering according to Chothia. In someembodiments, the amino acid substitution at position 1 is glutamic acid.In some embodiments, the amino acid substitution at position 3 isvaline. In some embodiments, the amino acid substitution at position 4is leucine. In some embodiments, the amino acid substitution at position9 is alanine. In some embodiments, the amino acid substitution atposition 10 is threonine. In some embodiments, the amino acidsubstitution at position 13 is leucine. In some embodiments, the aminoacid substitution at position 15 is proline. In some embodiments, theamino acid substitution at position 17 is glutamic acid. In someembodiments, the amino acid substitution at position 19 is alanine. Insome embodiments, the amino acid substitution at position 21 is leucine.In some embodiments, the amino acid substitution at position 22 isserine. In some embodiments, the amino acid substitution at position 38is histidine. In some embodiments, the amino acid substitution atposition 42 is glutamine. In some embodiments, the amino acidsubstitution at position 45 is arginine. In some embodiments, the aminoacid substitution at position 58 is isoleucine. In some embodiments, theamino acid substitution at position 60 is alanine. In some embodiments,the amino acid substitution at position 76 is threonine. In someembodiments, the amino acid substitution at position 77 is arginine. Insome embodiments, the amino acid substitution at position 77 isthreonine. In some embodiments, the amino acid substitution at position79 is glutamic acid. In some embodiments, the amino acid substitution atposition 85 is valine. In some embodiments, the amino acid substitutionat position 104 is leucine.

In another embodiment, the variable region sequences, or portionsthereof, of the anti-Zika virus EP antibodies described herein aremodified to create structurally related anti-Zika virus EP antibodiesthat retain binding (i.e., to the same epitope as the unmodifiedantibody).

Accordingly, in one aspect of the disclosure, the CDR1, 2, and/or 3regions of the engineered antibodies described above can comprise theexact amino acid sequence(s) as those of antibodies disclosed herein.However, in other aspects of the disclosure, the antibodies comprisederivatives from the exact CDR sequences of the antibodies disclosedherein, still retain the ability of to bind Zika virus EP effectively.Such sequence modifications may include one or more amino acidadditions, deletions, or substitutions, e.g., conservative sequencemodifications as described above. Sequence modifications may also bebased on the consensus sequences described above for the particularCDR1, CDR2, and CDR3 sequences of antibodies disclosed herein.

Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDRs that are, for example, 90%, 95%, 98% or99.5% identical to one or more CDRs of antibodies disclosed herein.Ranges intermediate to the above-recited values, e.g., CDRs that are90-95%, 95-98%, or 98-100% identical identity to one or more of theabove sequences are also intended to be encompassed by the presentdisclosure.

In another embodiment, one or more residues of a CDR may be altered tomodify binding to achieve a more favored on-rate of binding, a morefavored off-rate of binding, or both, such that an idealized bindingconstant is achieved. Using this strategy, an antibody having ultra-highbinding affinity of, for example, 10¹⁰ M⁻¹ or more, can be achieved.Affinity maturation techniques, well known in the art and thosedescribed herein, can be used to alter the CDR region(s) followed byscreening of the resultant binding molecules for the desired change inbinding. Accordingly, as CDR(s) are altered, changes in binding affinityas well as immunogenicity can be monitored and scored such that anantibody optimized for the best combined binding and low immunogenicityare achieved.

Thus, for variable region modification within the VH and/or VL CDR1,CDR2 and/or CDR3 regions, site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays. Preferably conservativemodifications (as discussed herein) are introduced. The mutations can beamino acid substitutions, additions or deletions, but are preferablysubstitutions. Moreover, typically no more than one, two, three, four orfive residues within a CDR region are altered.

In general, the framework regions of antibodies are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human germline sequences from which they were derived. Ofcourse, many of the amino acids in the framework region make little orno direct contribution to the specificity or affinity of an antibody.Thus, many individual conservative substitutions of framework residuescan be tolerated without appreciable change of the specificity oraffinity of the resulting immunoglobulin. Thus, in one embodiment thevariable framework region of the antibody shares at least 85% sequenceidentity to a human germline variable framework region sequence orconsensus of such sequences. In another embodiment, the variableframework region of the antibody shares at least 90%, 95%, 96%, 97%, 98%or 99% sequence identity to a human germline variable framework regionsequence or consensus of such sequences.

Framework modifications can also be made to reduce immunogenicity of theantibody or to reduce or remove T cell epitopes that reside therein, asdescribed for instance by Carr et al in US2003/0153043.

Engineered antibodies of the disclosure include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “back-mutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation cancontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino-terminal portion offramework region. For example, somatic mutations are relativelyinfrequent in the amino terminal portion of framework region 1 and inthe carboxy-terminal portion of framework region 4. Furthermore, manysomatic mutations do not significantly alter the binding properties ofthe antibody. For this reason, it is not necessary to obtain the entireDNA sequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see PCT/US99/05535 filed on Mar. 12, 1999). Partialheavy and light chain sequence spanning the CDR regions is typicallysufficient for this purpose. The partial sequence is used to determinewhich germline variable and joining gene segments contributed to therecombined antibody variable genes. The germline sequence is then usedto fill in missing portions of the variable regions. Heavy and lightchain leader sequences are cleaved during protein maturation and do notcontribute to the properties of the final antibody. To add missingsequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone. This process hascertain advantages such as elimination or inclusion or particularrestriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts from ahybridoma are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266:19867-19870); and, HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region (including the BbsI site of the kappa light chain, orthe AgeI site if the gamma heavy chain) in the PCR amplification togenerate fragments that can easily be cloned into the expression vectorconstructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader sequence, translation initiation,leader sequence, constant region, 3′ untranslated, polyadenylation, andtranscription termination, sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains.

Plasmids for use in construction of expression vectors were constructedso that PCR amplified V heavy and V kappa light chain cDNA sequencescould be used to reconstruct complete heavy and light chain minigenes.These plasmids can be used to express completely human IgG_(1κ) orIgG_(4κ) antibodies.

Fully human, humanized, and chimeric antibodies described herein alsoinclude IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar plasmidscan be constructed for expression of other heavy chain isotypes, or forexpression of antibodies comprising lambda light chains.

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 20, 26 and 31; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 38, 44 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 22, 28 and 33; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 39, 45 and51; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 20, 26 and 31; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 39, 45 and51; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 20, 26 and 31; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 40, 46 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 28 and 32; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 38, 44 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 27 and 32; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 40, 46 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 27 and 32; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 38, 44 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 27 and 32; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 37, 43 and49; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 28 and 32; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 37, 43 and49; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 22, 28 and 33; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 37, 43 and49; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 23, 28 and 34; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 37, 43 and49; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 28 and 34; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 37, 43 and49; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 27 and 32; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 39, 45 and51; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 28 and 32; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 39, 45 and51; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 28 and 32; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 40, 46 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 22, 28 and 33; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 38, 44 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 22, 28 and 33; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 40, 46 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 23, 28 and 34; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 38, 44 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 23, 28 and 34; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 40, 46 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 28 and 34; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 38, 44 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 28 and 34; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 39, 45 and51; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

In certain embodiments, methods for preparing an anti-Zika virus EPantibody are provided, including: preparing an antibody including (1)heavy chain framework regions and heavy chain CDRs, where at least oneof the heavy chain CDRs includes an amino acid sequence selected fromthe amino acid sequences of CDRs shown in SEQ ID NOs: 21, 28 and 34; and(2) light chain framework regions and light chain CDRs, where at leastone of the light chain CDRs includes an amino acid sequence selectedfrom the amino acid sequences of CDRs shown in SEQ ID NOs: 40, 46 and50; where the antibody binds to Zika virus EP. The ability of theantibody to bind Zika virus EP can be determined using standard bindingassays (e.g., an ELISA or a FLISA).

Additional Antibody Modifications

Antibodies of the present disclosure can contain one or moreglycosylation sites in either the light or heavy chain variable region.Such glycosylation sites may result in increased immunogenicity of theantibody or an alteration of the pK of the antibody due to alteredantigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Galaand Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J ExpMed 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al(1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. In some instances, it is preferred to have an anti-Zika virusantibody that does not contain variable region glycosylation. This canbe achieved either by selecting antibodies that do not contain theglycosylation motif in the variable region or by mutating residueswithin the glycosylation region.

For example, in certain embodiments, the glycosylation of an antibody ismodified, e.g., the variable region is altered to eliminate one or moreglycosylation sites resident in the variable region. More particularly,it is desirable in the sequence of the present antibodies to eliminatesites prone to glycosylation. This is achieved by altering theoccurrence of one or more N-X-(S/T) sequences that occur in the parentvariable region (where X is any amino acid residue), particularly bysubstituting the N residue and/or the S or T residue. In one embodiment,T95 is mutated to K95. In another embodiment, N47 is mutated to R47.

For example, aglycoslated antibodies can be made (i.e., which lackglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos.5,714,350 and 6,350,861.

Additionally or alternatively, the antibody can have an altered type ofglycosylation, such as a hypofucosylated antibody having reduced amountsof fucosyl residues or an antibody having increased bisecting GlcNacstructures. Such altered glycosylation patterns have been demonstratedto increase the ADCC ability of antibodies. Such carbohydratemodifications can be accomplished by, for example, expressing theantibody in a host cell with altered glycosylation machinery. Cells withaltered glycosylation machinery have been described in the art and canbe used as host cells in which to express recombinant antibodiesdescribed herein to thereby produce an antibody with alteredglycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lackthe fucosyltransferase gene, FUT8 (α (1,6)-fucosyltransferase), suchthat antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8^(−/−)cell lines were created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 and Yamane-Ohnuki et al. (2004) BiotechnolBioeng 87:614-22). As another example, EP 1,176,195 describes a cellline with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the α-1,6 bond-relatedenzyme. EP 1,176,195 also describes cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFc region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 describes a variant CHO cell line, Lec13 cells, withreduced ability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740).Antibodies with a modified glycosylation profile can also be produced inchicken eggs, as described in PCT Publication WO 06/089231.Alternatively, antibodies with a modified glycosylation profile can beproduced in plant cells, such as Lemna. Methods for production ofantibodies in a plant system are disclosed in the U.S. Patentapplication corresponding to Alston & Bird LLP attorney docket No.040989/314911, filed on Aug. 11, 2006. PCT Publication WO 99/54342describes cell lines engineered to express glycoprotein-modifyingglycosyl transferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III(GnTIII)) such that antibodies expressed in the engineered cell linesexhibit increased bisecting GlcNac structures which results in increasedADCC activity of the antibodies (see also Umana et al. (1999) Nat.Biotech. 17:176-180). Alternatively, the fucose residues of the antibodycan be cleaved off using a fucosidase enzyme; e.g., the fucosidaseα-L-fucosidase removes fucosyl residues from antibodies (Tarentino etal. (1975) Biochem. 14:5516-23).

The variable segments of antibodies produced as described supra (e.g.,the heavy and light chain variable regions of chimeric or humanizedantibodies) are typically linked to at least a portion of animmunoglobulin constant region (Fc region), typically that of a humanimmunoglobulin. Human constant region DNA sequences can be isolated inaccordance with well-known procedures from a variety of human cells, butpreferably immortalized B cells (see Kabat et al., supra, and Liu etal., WO87/02671) (each of which is incorporated by reference in itsentirety for all purposes). Ordinarily, the antibody will contain bothlight chain and heavy chain constant regions. The heavy chain constantregion usually includes CH1, hinge, CH2, CH3, and CH4 regions. Theantibodies described herein include antibodies having all types ofconstant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG1, IgG2, IgG3 and IgG4. When it is desired that theantibody (e.g., humanized antibody) exhibit cytotoxic activity, theconstant domain is usually a complement fixing constant domain and theclass is typically IgG1. Human isotype IgG1 is preferred. Light chainconstant regions can be lambda or kappa. The humanized antibody maycomprise sequences from more than one class or isotype. Antibodies canbe expressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′F(ab′)2, and Fv, or assingle chain antibodies in which heavy and light chain variable domainsare linked through a spacer.

In certain embodiments, the antibody comprises a variable region that ismutated to improve the physical stability of the antibody. In oneembodiment, the antibody is an IgG4 isotype antibody comprising a serineto proline mutation at a position corresponding to position 228 (S228P;EU index) in the hinge region of the heavy chain constant region. Thismutation has been reported to abolish the heterogeneity of inter-heavychain disulfide bridges in the hinge region (Angal et al. supra;position 241 is based on the Kabat numbering system). For example, incertain embodiments, an anti-Zika virus EP antibody described herein cancomprise the heavy chain variable region of any of the antibodiesdescribed herein linked to a human IgG4 constant region in which theSerine at a position corresponding to position 241 as described in Angalet al., supra, has been mutated to Proline. Thus, for the heavy chainvariable regions linked to a human IgG4 constant region, this mutationcorresponds to an S228P mutation by the EU index.

In certain embodiments, the hinge region of CH1 is modified such thatthe number of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In addition, the antibody can be pegylated, for example, to increase thebiological (e.g., serum) half-life of the antibody. To pegylate anantibody, the antibody, or fragment thereof, typically is reacted withpolyethylene glycol (PEG), such as a reactive ester or aldehydederivative of PEG, under conditions in which one or more PEG groupsbecome attached to the antibody or antibody fragment. Preferably, thepegylation is carried out via an acylation reaction or an alkylationreaction with a reactive PEG molecule (or an analogous reactivewater-soluble polymer). As used herein, the term “polyethylene glycol”is intended to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies described herein. See, e.g., EP 0 154 316 and EP 0 401384.

Immunizations

To generate fully human antibodies to Zika virus EP, transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) can be immunized with a purified or enrichedpreparation of the Zika virus EP antigen and/or cells expressing Zikavirus EP, as described, for example, by Lonberg et al. (1994) Nature368(6474): 856-859; Fishwild et al. (1996) Nature Biotechnology 14:845-851 and WO 98/24884. As described herein, HuMAb mice are immunizedeither with recombinant Zika virus EP proteins or cell lines expressingZika virus EP as immunogens. Alternatively, mice can be immunized withDNA encoding Zika virus EP. Preferably, the mice will be 6-16 weeks ofage upon the first infusion. For example, a purified or enrichedpreparation (5-50 μg) of the recombinant Zika virus EP antigen can beused to immunize the HuMAb mice intraperitoneally.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) or subcutaneously (SC) with antigen in complete Freund's adjuvant,followed by every other week IP/SC immunizations (up to a total of 10)with antigen in incomplete Freund's adjuvant. The immune response can bemonitored over the course of the immunization protocol with plasmasamples being obtained by retroorbital bleeds. The plasma can bescreened by ELISA (as described below), and mice with sufficient titersof anti-Zika virus EP human immunoglobulin can be used for fusions. Micecan be boosted intravenously with antigen 3 days before sacrifice andremoval of the spleen.

Generation of Hybridomas Producing Monoclonal Antibodies to Zika VirusEP

To generate hybridomas producing monoclonal antibodies to Zika virus EP,splenocytes and lymph node cells from immunized mice can be isolated andfused to an appropriate immortalized cell line, such as a mouse myelomacell line. The resulting hybridomas can then be screened for theproduction of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toSP2/0-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50%PEG (w/v). Cells can be plated at approximately 1×10⁵ in flat bottommicrotiter plate, followed by a two week incubation in selective mediumcontaining besides usual reagents 10% fetal Clone Serum, 5-10% origenhybridoma cloning factor (IGEN) and 1×HAT (Sigma). After approximatelytwo weeks, cells can be cultured in medium in which the HAT is replacedwith HT. Individual wells can then be screened by ELISA for humananti-Zika virus EP monoclonal IgM and IgG antibodies. Once extensivehybridoma growth occurs, medium can be observed usually after 10-14days. The antibody secreting hybridomas can be replated, screened again,and if still positive for IgG, anti-Zika virus EP monoclonal antibodiescan be subcloned at least twice by limiting dilution. The stablesubclones can then be cultured in vitro to generate antibody in tissueculture medium for characterization.

Generation of Transfectomas Producing Monoclonal Antibodies to ZikaVirus EP

Antibodies described herein also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S. (1985) Science 229:1202).

For example, in certain embodiments, the gene(s) of interest, e.g.,human antibody genes, can be ligated into an expression vector such as aeukaryotic expression plasmid such as used by GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such asCHO-cells or NSO-cells or alternatively other eukaryotic cells like aplant derived cells, fungi or yeast cells. The method used to introducethese genes could be methods described in the art such aselectroporation, lipofectine, lipofectamine or other. After introducingthese antibody genes in the host cells, cells expressing the antibodycan be identified and selected. These cells represent the transfectomaswhich can then be amplified for their expression level and upscaled toproduce antibodies. Recombinant antibodies can be isolated and purifiedfrom these culture supernatants and/or cells.

Alternatively these cloned antibody genes can be expressed in otherexpression systems such as E. coli or in complete organisms or can besynthetically expressed.

Expression of Recombinant Antibodies

Chimeric and humanized antibodies are typically produced by recombinantexpression. Nucleic acids encoding light and heavy chain variableregions, optionally linked to constant regions, are inserted intoexpression vectors. The light and heavy chains can be cloned in the sameor different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. Preferably, the expression control sequences are eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences, and the collectionand purification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance,kanamycin resistance or neomycin resistance) to permit detection ofthose cells transformed with the desired DNA sequences (see, e.g.,Itakura et al., U.S. Pat. No. 4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) described herein. Other microbialhosts suitable for use include bacilli, such as Bacillus subtilis, andother enterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation. Other microbes, such as yeast, are alsouseful for expression.

Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of described herein (e.g.,polynucleotides encoding immunoglobulins or fragments thereof). SeeWinnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various Cos cell lines, HeLa cells, preferably, myeloma celllines, or transformed B-cells or hybridomas. Preferably, the cells arenonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment, electroporation, lipofection,biolistics or viral-based transfection may be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated byreference in its entirety for all purposes). Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole antibodies, theirdimers, individual light and heavy chains, or other immunoglobulin formsof the present disclosure can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses.

Antibody Fragments

Also contemplated within the scope of the instant disclosure areantibody fragments. In one embodiment, fragments of non-human, and/orchimeric antibodies are provided. In another embodiment, fragments ofhumanized antibodies are provided. Typically, these fragments exhibitspecific binding to antigen with an affinity of at least 10⁷, and moretypically 10⁸ or 10⁹ M⁻¹. Humanized antibody fragments include separateheavy chains, light chains, Fab, Fab′, F(ab′)2, Fabc, and Fv. Fragmentsare produced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins.

Assays for Characterization of Antibodies

Antibodies described herein can be tested for binding to Zika virusenvelope protein (EP) by, for example, standard ELISA. Briefly, serialdilutions of Zika virus envelope protein, or Zika virus itself, is mixedwith antibodies described herein. These mixtures are incubated overnightat room temperature to allow equilibrium to be reached. An indirectELISA is used to measure the concentration of unbound and singly boundantibody. Alternatively, microtiter plates are coated with purified Zikavirus and then blocked with 5% nonfat dry milk in PBS. After washing,purified antibodies described herein are added to wells containingantigen and incubated for 2 hours at room temperature. Bound antibodiesare detected using horseradish peroxidase conjugated anti-mouse IgGsecondary antibodies and ABTS substrate (Kirkegaard and PerryLaboratories).

In some embodiments, the antibodies described herein bind Zika virus EPwith a Kd value between 3 and 25 μg/mL as determined by ELISA. In someembodiments, the antibodies described herein bind Zika virus EP with aKd value of at least 3 μg/mL as determined by ELISA. In someembodiments, the antibodies described herein bind Zika virus EP with aKd value of at least 5 μg/mL as determined by ELISA. In someembodiments, the antibodies described herein bind Zika virus EP with aKd value of at least 10 μg/mL as determined by ELISA. In someembodiments, the antibodies described herein bind Zika virus EP with aKd value of at least 15 μg/mL as determined by ELISA. In someembodiments, the antibodies described herein bind Zika virus EP with aKd value of at least 20 μg/mL as determined by ELISA. In someembodiments, the antibodies described herein bind Zika virus EP with aKd value of at least 25 μg/mL as determined by ELISA.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the Zika virus EP. Hybridomas that produce antibodiesthat bind, preferably with high affinity, to Zika virus EP can then besubcloned and further characterized. One clone from each hybridoma,which retains the reactivity of the parent cells (by ELISA), can then bechosen for making a cell bank, and for antibody purification.

The ELISA assay described above can also be used to confirm thatframework mutation(s) do not affect the ability of the anti-EPantibodies disclosed herein to bind to EP.

To purify anti-Zika virus EP antibodies, selected hybridomas can begrown in two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-Zika virus EP monoclonal antibodiesbind to unique epitopes, each antibody can be biotinylated usingcommercially available reagents (Pierce, Rockford, Ill.). BiotinylatedMAb binding can be detected with a streptavidin labeled probe.Competition studies using unlabeled monoclonal antibodies andbiotinylated monoclonal antibodies can be performed using Zika virus EPcoated-ELISA plates as described above.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, plates are coated with anti-IgG, IgA, or IgMheavy-chain specific antibodies (100 ng/well) and incubated withhybridoma culture supernatants. The subtype of the mAb is detected byusing anti-IgG1, IgG2a, IgG2b, IgG3, IgM, or IgA heavy-chain specificantibodies conjugated to alkaline phosphatase.

Anti-Zika virus EP antibodies can be further tested for reactivity withthe Zika virus EP antigen by Western blotting. Briefly, unlabeled Zikavirus EP is resolved on a 10% SDS-polyacrylaminde gel and transferred toPVDF membranes. After nonspecific bindings sites are blocked usingnonfat dry milk in PBS containing 0.02% Tween-20, purified mAb (10ug/ml) are added to the membranes for 1 hour at room temperature.Membranes are then incubated with horseradish peroxidase-conjugated goatanti-mouse IgA+IgG+IgM secondary antibodies for 1 hour and developedusing ECL chemiluminescence kit (Amersham).

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-Zika virus EP antibodies include standardassays known in the art, for example, Biacore™ surface plasmon resonance(SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB,Uppsala, Sweden).

To determine the neutralization of antibodies described herein, in vitroplaque reduction neutralization (PRNT) assays can be done. Briefly,plaque assays are done using confluent BHK-21 cells. Two-fold serialdilutions of antibodies described herein are mixed with 50 PFU of Zikavirus at 37° C. for 1 hour. The mixture is applied to cell monoloayersand incubated for 4-7 days. Infection is quantified by 4G2immunostaining followed by TMB peroxidase substrate (KPL) and absorbanceis measured in a plate reader. 100% infection corresponds to the averageOD600 in wells not exposed to antibody. In some embodiments, the outputof the PRNT assay is PRNT50 (concentration of antibody that reducesplaque formation by 50%). In some embodiments, the antibodies describedherein have a PRNT50 value of at least 0.03 μg/mL.

In some embodiments, anti-Zika virus EP antibodies reduceantibody-dependent enhancement (ADE) of infection. ADE is a phenomenonthat has been proposed to mediate increased disease severity wheninfection occurs in a background of preexisting enhancing antibodies.Mechanistically, this occurs when enhancing antibodies bind the matureas well as immature virus particles and mediate virus entry via antibodyengagement of Fcγ receptors present on host cells. Several FLE-directedantibodies have been described in literature including 4G2, E53, and theE-dimer epitope (EDE) directed mAbs (Dejnirattisai W, et al., 2015).Several studies have shown that when DENV is opsonized with antibodylevels that are phagocytosed by Fcγ receptors, only antibodies that areable to inhibit virus fusion with phagosomal membranes will preventinfection and thus ADE (Chan K R et al., 2011, Wu R et al, 2012).Accordingly, in some embodiments, the ability of the anti-Zika virus EPantibodies described herein to engage the Zika virus FLE epitope at theE-dimer interface, results in reduction of ADE activity by fusioninhibition.

In some embodiments, reduction of ADE is tested by using cells thatexpress leukocyte immunoglobulin like receptor B 1 (LILRB1) and are thushighly susceptible to ADE. A non-limiting example of such cells isTHP1.2S monocytes. Cells are incubated with an anti-Zika virus antibodydescribed herein prior to infection. Virus replication is then measuredby plaque assay using cells susceptible to viral infection. Anon-limiting example of such cells is BHK21 cells. Plaque titers arethen measured, and a reduction in plaque titers compared to a controlindicates the anti-Zika virus EP antibody tested is effective inreducing ADE. Other methods of measuring ADE are known in the art andcan be used to determine the effect of an anti-Zika virus EP antibodydescribed herein on ADE.

Competitive Binding Antibodies

In certain embodiments, antibodies described herein compete (e.g.,cross-compete) for binding to Zika virus EP with the particularanti-Zika virus EP antibodies described herein. Such competingantibodies can be identified based on their ability to competitivelyinhibit binding to Zika virus EP of one or more of mAbs described hereinin standard Zika virus EP binding assays. For example, standard ELISAassays can be used in which a recombinant Zika virus EP is immobilizedon the plate, one of the antibodies is fluorescently labeled and theability of non-labeled antibodies to compete off the binding of thelabeled antibody is evaluated. Additionally or alternatively, BIAcoreanalysis can be used to assess the ability of the antibodies tocross-compete. The ability of a test antibody to inhibit the binding ofan anti-EP antibody described herein to Zika virus EP demonstrates thatthe test antibody can compete with the antibody for binding to Zikavirus EP.

In certain embodiments, the competing antibody is an antibody that bindsto the same epitope on Zika virus EP as the particular anti-EPmonoclonal antibodies described herein. Standard epitope mappingtechniques, such as x-ray crystallography and 2-dimensional nuclearmagnetic resonance, can be used to determine whether an antibody bindsto the same epitope as a reference antibody (see, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.(1996)).

In certain embodiments, the antibody that competes for binding to Zikavirus EP and/or binds to the same epitope on Zika virus EP is ahumanized antibody.

In some embodiments, the anti-Zika virus antibodies described hereinbind an epitope on the fusion loop comprising residues D98, R99 and W101(SEQ ID NO: 3). In some embodiments, the antibody that competes forbinding to Zika virus EP binds the epitope on the fusion loop comprisingresidues D98, R99 and W101 (SEQ ID NO: 3).

Once a single, archtypal anti-EP mAb has been isolated that has thedesired properties described herein, it is straightforward to generateother mAbs with similar properties, e.g., having the same epitope, byusing art-known methods. For example, mice may be immunized with Zikavirus as described herein, hybridomas produced, and the resulting mAbsscreened for the ability to compete with the archtypal mAb for bindingto Zika virus EP. Mice can also be immunized with a smaller fragment ofZika virus EP containing the epitope to which the archtypal mAb binds.The epitope can be localized by, e.g., screening for binding to a seriesof overlapping peptides spanning Zika virus EP. Alternatively, themethod of Jespers et al., Biotechnology 12:899, 1994 may be used toguide the selection of mAbs having the same epitope and thereforesimilar properties to the archtypal mAb. Using phage display, first theheavy chain of the archtypal antibody is paired with a repertoire of(preferably human) light chains to select an Zika virus EP-binding mAb,and then the new light chain is paired with a repertoire of (preferablyhuman) heavy chains to select an (preferably human) Zika virusEP-binding mAb having the same epitope as the archtypal mAb.Alternatively variants of the archetypal mAb can be obtained bymutagenesis of cDNA encoding the heavy and light chains of the antibody.

Epitope mapping, e.g., as described in Champe et al. (1995) J. Biol.Chem. 270:1388-1394, can be performed to determine whether the antibodybinds an epitope of interest. “Alanine scanning mutagenesis,” asdescribed by Cunningham and Wells (1989) Science 244: 1081-1085, or someother form of point mutagenesis of amino acid residues in human Zikavirus EP may also be used to determine the functional epitope for ananti-EP antibody described herein. Mutagenesis studies, however, mayalso reveal amino acid residues that are crucial to the overallthree-dimensional structure of Zika virus EP but that are not directlyinvolved in antibody-antigen contacts, and thus other methods may benecessary to confirm a functional epitope determined using this method.

The epitope bound by a specific antibody may also be determined byassessing binding of the antibody to peptides comprising fragments ofZika virus EP. A series of overlapping peptides encompassing thesequence of Zika virus EP may be synthesized and screened for binding,e.g. in a direct ELISA, a competitive ELISA (where the peptide isassessed for its ability to prevent binding of an antibody to Zika virusEP bound to a well of a microtiter plate), or on a chip. Such peptidescreening methods may not be capable of detecting some discontinuousfunctional epitopes, i.e. functional epitopes that involve amino acidresidues that are not contiguous along the primary sequence of the Zikavirus EP polypeptide chain.

The epitope bound by antibodies described herein may also be determinedby structural methods, such as X-ray crystal structure determination(e.g., WO2005/044853), molecular modeling and nuclear magnetic resonance(NMR) spectroscopy, including NMR determination of the H-D exchangerates of labile amide hydrogens in Zika virus EP when free and whenbound in a complex with an antibody of interest (Zinn-Justin et al.(1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993)Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J.Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLand preferably about 10 mg/mL to about 20 mg/mL. Crystallization may bebest achieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about3.0 to about 5.0, preferably about 4.0. Specific buffers useful in theprecipitant solution may vary and are well-known in the art (Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals may be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody:antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al., eds., Academic Press; U.S. Patent ApplicationPublication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst.D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet,eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosuresof which are hereby incorporated by reference in their entireties.

Antibody competition assays, as described herein, can be used todetermine whether an antibody “binds to the same epitope” as anotherantibody. Typically, competition of 50% or more, 60% or more, 70% ormore, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more,of an antibody known to interact with the epitope by a second antibodyunder conditions in which the second antibody is in excess and the firstsaturates all sites, is indicative that the antibodies “bind to the sameepitope.” To assess the level of competition between two antibodies, forexample, radioimmunoassays or assays using other labels for theantibodies, can be used. For example, a Zika virus EP antigen can beincubated with a saturating amount of a first anti-EP antibody orantigen-binding fragment thereof conjugated to a labeled compound (e.g.,³H, ¹²⁵I, biotin, or rubidium) in the presence the same amount of asecond unlabeled anti-EP antibody. The amount of labeled antibody thatis bound to the antigen in the presence of the unlabeled blockingantibody is then assessed and compared to binding in the absence of theunlabeled blocking antibody. Competition is determined by the percentagechange in binding signals in the presence of the unlabeled blockingantibody compared to the absence of the blocking antibody. Thus, ifthere is a 50% inhibition of binding of the labeled antibody in thepresence of the blocking antibody compared to binding in the absence ofthe blocking antibody, then there is competition between the twoantibodies of 50%/a. Thus, reference to competition between a first andsecond antibody of 50% or more, 60% or more, 70% or more, such as 70%,71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more, means that thefirst antibody inhibits binding of the second antibody (or vice versa)to the antigen by 50%, 60%/a, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%,90%, 95% or more (compared to binding of the antigen by the secondantibody in the absence of the first antibody). Thus, inhibition ofbinding of a first antibody to an antigen by a second antibody of 50%,60%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more indicatesthat the two antibodies bind to the same epitope.

Testing Antibodies for Therapeutic Efficacy in Animal Models

Animal models are effective for testing the therapeutic efficacy ofantibodies against Zika virus EP. In certain embodiments, rodents (i.e.,mice) can be used for Zika infection. Briefly, mice are challenged witha strain of mouse adapted Zika virus (e.g., H/PF/2013 (French Polynesia2013)) by intraperitoneal inoculation, approximately 300 times the doselethal for 50% of adult mice. In certain embodiments, guinea pigs can beused for Zika infection. Guinea pigs are challenged with guineapig-adapted virus. Additionally, in certain embodiments, non-humanprimates are used as an animal model of infection. In all animal models,antibodies can be administered 24 or 48 hours after infection to testfor therapeutic efficacy. In some embodiments, antibodies areadministered 24 or 48 hours before infection to test for prophylacticefficacy.

Efficacy of the antibodies to prevent or treat Zika virus is determinedby analyzing the mortality rate, viral load, and/or body weight of theanimals over time. In some embodiments, untreated animals infected withZika virus die within 10-11 days post infection. In some embodiments,animals treated with the anti-Zika antibodies described herein before orafter Zika virus infection, live significantly longer compared tountreated animals. In some embodiments, animals treated with theanti-Zika antibodies described herein before or after Zika virusinfection, live up to 22 days. In some embodiments, animals treated withthe anti-Zika antibodies described herein before or after Zika virusinfection, maintain body weight over time compared to untreated animals.In some embodiments, animals treated with the anti-Zika antibodiesdescribed herein before or after Zika virus infection, have a reducedviral load compared to untreated animals.

In some embodiments, animal models (e.g., mice) are used to determinethe efficacy of an anti-Zika virus EP antibody in treating or preventingvertical infection and fetal mortality in pregnancy. In someembodiments, animal models are used to determine the efficacy of ananti-Zika virus EP antibody in reducing or reducing the risk of verticalinfection and fetal mortality in pregnancy. In some embodiments,pregnant animals are infected with an adapted Zika virus (e.g.,H/PF/2013 for mice) intravenously. Anti-Zika virus EP antibodiesdescribed herein are administered before or after infection to test fortherapeutic or prophylactic efficacy, respectively. In some embodiments,the efficacy of the antibodies is determined by measuring the viral loadin the mother, fetus and placenta, along with determining the fetussurvival rate. In some embodiments, mothers treated with the anti-Zikaantibodies described herein have a reduced viral load. In someembodiments, fetuses from mothers treated with the anti-Zika antibodiesdescribed herein have a higher survival rate and reduced viral load. Insome embodiments, the higher survival rate of fetuses is measured asreduction in percent lethality. In some embodiments, the placenta frommothers treated with the anti-Zika antibodies described herein has areduced viral load. In some embodiments, fetuses from mothers treatedwith the anti-Zika virus antibodies described herein have normal embryodevelopment. In some embodiments, fetuses from mothers treated with theanti-Zika virus antibodies described herein have normal embryodevelopment, compared to untreated mothers. In some embodiments, thefetuses from untreated mothers are dead (i.e., 100% lethality).

Immunotoxins, Immunoconjugates and Antibody Derivatives

In another embodiment, the antibodies described herein are linked to atherapeutic moiety, such as a cytotoxin, a drug or a radioisotope. Whenconjugated to a cytotoxin, these antibody conjugates are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells.

Techniques for conjugating such therapeutic moiety to antibodies arewell known in the art.

The toxin component of the immunotoxin can be, for example, achemotherapeutic agent, a toxin such as an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof, or asmall molecule toxin.

Additional toxins and fragments thereof which can be used includediphtheria A chain, nonbonding active fragments of diphtheria toxin,cholera toxin, botulinus toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolacaAmericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, sapaonaria, officinalis inhibitor, gelonin,saporin, mitogellin, restrictocin, phenomycin, enomycin, and thetricothcenes. Small molecule toxins include, for example,calicheamicins, maytansinoids, palytoxin and CC1065.

Antibodies described herein also can be used for diagnostic purposes,including sample testing and in vivo imaging, and for this purpose theantibody (or binding fragment thereof) can be conjugated to anappropriate detectable agent, to form an immunoconjugate. For diagnosticpurposes, appropriate agents are detectable labels that includeradioisotopes, for whole body imaging, and radioisotopes, enzymes,fluorescent labels and other suitable antibody tags for sample testing.

For Zika virus EP detection, the detectable labels can be any of thevarious types used currently in the field of in vitro diagnostics,including particulate labels including metal sols such as colloidalgold, isotopes such as I¹²⁵ or Tc⁹⁹ presented for instance with apeptidic chelating agent of the N₂S₂, N₃S or N₄ type, chromophoresincluding fluorescent markers, luminescent markers, phosphorescentmarkers and the like, as well as enzyme labels that convert a givensubstrate to a detectable marker, and polynucleotide tags that arerevealed following amplification such as by polymerase chain reaction.Suitable enzyme labels include horseradish peroxidase, alkalinephosphatase and the like. For instance, the label can be the enzymealkaline phosphatase, detected by measuring the presence or formation ofchemiluminescence following conversion of 1,2 dioxetane substrates suchas adamantyl methoxy phosphoryloxy phenyl dioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-Star®or other luminescent substrates well-known to those in the art, forexample the chelates of suitable lanthanides such as Terbium(III) andEuropium(III). The detection means is determined by the chosen label.Appearance of the label or its reaction products can be achieved usingthe naked eye, in the case where the label is particulate andaccumulates at appropriate levels, or using instruments such as aspectrophotometer, a luminometer, a fluorimeter, and the like, all inaccordance with standard practice.

In certain embodiments, an antibody provided herein may be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

Compositions

In certain embodiments, a composition, e.g., a composition, containingone or more monoclonal antibodies described herein, formulated togetherwith a carrier (e.g., a pharmaceutically acceptable carrier), isprovided. In some embodiments, the compositions include a combination ofmultiple (e.g., two or more) isolated antibodies described herein.Preferably, each of the antibodies of the composition binds to adistinct, pre-selected epitope of Zika virus EP.

Pharmaceutical compositions described herein also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition described herein with atleast one or more additional therapeutic agents. Co-administration withother antibodies is also encompassed by the disclosure.

As used herein, the terms “carrier” and “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Preferably,the carrier is suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g., by injection orinfusion). Depending on the route of administration, the activecompound, e.g., antibody, may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition described herein can be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. The active compounds can be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound described herein by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent. Acceptablediluents include saline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al. (1984) J. Neuroimmunol. 7:27).

Carriers include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the activecompound, use thereof in the pharmaceutical compositions describedherein is contemplated. Supplementary active compounds can also beincorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, the antibodiesdescribed herein may be administered once or twice weekly bysubcutaneous or intramuscular injection or once or twice monthly bysubcutaneous or intramuscular injection.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit formsdescribed herein are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations described herein includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.001 percent to about ninetypercent of active ingredient, preferably from about 0.005 percent toabout 70 percent, most preferably from about 0.01 percent to about 30percent.

Formulations described herein which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions described herein include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions described herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds described herein are administered as pharmaceuticals,to humans and animals, they can be given alone or as a pharmaceuticalcomposition containing, for example, 0.001 to 90% (more preferably,0.005 to 70%, such as 0.01 to 30%) of active ingredient in combinationwith a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compoundsdescribed herein, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions described herein, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions described herein employed,or the ester, salt or amide thereof, the route of administration, thetime of administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular compositionsemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts. A physician or veterinarian having ordinaryskill in the art can readily determine and prescribe the effectiveamount of the pharmaceutical composition required. For example, thephysician or veterinarian could start doses of the compounds describedherein employed in the pharmaceutical composition at levels lower thanthat required in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, a suitable daily dose of a composition described herein will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. It is preferred thatadministration be intravenous, intramuscular, intraperitoneal, orsubcutaneous, preferably administered proximal to the site of thetarget. If desired, the effective daily dose of a therapeuticcomposition may be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms. While it is possible for acompound described herein to be administered alone, it is preferable toadminister the compound as a pharmaceutical formulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in certain embodiments, a therapeuticcomposition described herein can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,or 4,596,556. Examples of well-known implants and modules useful in thepresent disclosure include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the antibodies described herein can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds described herein cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations describedherein, as well as components of the invented molecules; p120 (Schreieret al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L.Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994)Immunomethods 4:273. In one embodiment, the therapeutic compoundsdescribed herein are formulated in liposomes; in certain embodiments,the liposomes include a targeting moiety. In certain embodiments, thetherapeutic compounds in the liposomes are delivered by bolus injectionto a site proximal to the tumor or infection. The composition must befluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

Uses and Methods

In certain embodiments, the antibodies, bispecific molecules, andcompositions described herein can be used to treat and/or prevent (e.g.,immunize against) Zika virus infection. The ability of the antibodiesdescribed herein to bind the fusion loop within domain II of theenvelope protein, suggests cross reactivity within the flavivirusfamily. Therefore, in some embodiments, the antibodies, bispecificmolecules and compositions described herein can be used to treat and/orprevent (e.g., immunize against) any member of the flavivirus family.

For use in therapy, the antibodies described herein can be administeredto a subject directly (i.e., in vivo), either alone or with othertherapies such as an immunostimulatory agent. In all cases, theantibodies, compositions, and immunostimulatory agents and othertherapies are administered in an effective amount to exert their desiredtherapeutic effect. The term “effective amount” refers to that amountnecessary or sufficient to realize a desired biologic effect. One ofordinary skill in the art can empirically determine the effective amountof a particular molecule without necessitating undue experimentation.

Preferred routes of administration for vaccines include, for example,injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal,intrathecal). The injection can be in a bolus or a continuous infusion.Other routes of administration include oral administration.

Antibodies described herein also can be coadministered with adjuvantsand other therapeutic agents. It will be appreciated that the term“coadministered” as used herein includes any or all of simultaneous,separate, or sequential administration of the antibodies and conjugatesdescribed herein with adjuvants and other agents, includingadministration as part of a dosing regimen. The antibodies are typicallyformulated in a carrier alone or in combination with such agents.Examples of such carriers include solutions, solvents, dispersion media,delay agents, emulsions and the like. The use of such media forpharmaceutically active substances is well known in the art. Any otherconventional carrier suitable for use with the molecules falls withinthe scope of the instant disclosure.

In certain embodiments, the antibodies described herein can be utilizedfor prophylactic applications. In certain embodiments, prophylacticapplications involve systems and methods for preventing, inhibitingprogression of, and/or delaying the onset of Zika virus infection,and/or any other Zika virus-associated condition in individualssusceptible to and/or displaying symptoms of Zika virus infection. Incertain embodiments, prophylactic applications involve systems andmethods for preventing, inhibiting progression of, and/or delaying thedevelopment of microcephaly in newborn babies of mothers with Zika virusinfection.

In some embodiments, the antibodies described herein are utilized fortreating or preventing vertical transmission of Zika virus infection ina pregnant subject. In some embodiments, the antibodies described hereinare utilized for reducing or reducing the risk of vertical transmissionof Zika virus infection in a pregnant subject. In some embodiments, theantibodies are administered to a pregnant subject infected with Zikavirus. In some embodiments, the antibodies are administered to apregnant subject at risk of being infected with Zika virus. In someembodiments, the antibodies described herein are utilized for treatingor preventing fetal mortality in a pregnant subject infected with Zikavirus or at risk of being infected with Zika virus. In some embodiments,the antibodies described herein are utilized for reducing or reducingthe risk of fetal mortality in a pregnant subject infected with Zikavirus or at risk of being infected with Zika virus. In some embodiments,the antibodies described herein are utilized to reduce viral load in apregnant subject, the fetus, and/or the placenta, wherein the pregnantsubject is infected with Zika virus. Viral load can be measured asdescribed herein.

Peptides and Compositions Based on Zika Virus Envelope Protein (EP)Epitopes

The antibodies described above are formulated into vaccine compositions.These vaccine compositions may be employed to immunize a subject inorder to elicit a highly anti-Zika antibody immune response. Vaccinecompositions are also useful to administer to subjects in need thereofto induce a protective immune response. Such vaccine compositions arewell known in the art and include, for example, physiologicallycompatible buffers, preservatives, and saline and the like, as welladjuvants.

“Adjuvants” are agents that nonspecifically increase an immune responseto a particular antigen, thus reducing the quantity of antigen necessaryin any given vaccine, and/or the frequency of injection necessary inorder to generate an adequate immune response to the antigen ofinterest. Suitable adjuvants for the vaccination of animals include, butare not limited to, Adjuvant 65 (containing peanut oil, mannidemonooleate and aluminum monostearate); Freund's complete or incompleteadjuvant; mineral gels, such as aluminum hydroxide, aluminum phosphateand alum; surfactants, such as hexadecylamine, octadecylamine,lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecylN′,N′-bis(2-hydroxymethyl) propanediamine, methoxyhexadecylglycerol andpluronic polyols; polyanions, such as pyran, dextran sulfate, poly IC,polyacrylic acid and carbopol; peptides, such as muramyl dipeptide,dimethylglycine and tuftsin; and oil emulsions. The protein or peptidescould also be administered following incorporation into liposomes orother microcarriers. Information concerning adjuvants and variousaspects of immunoassays are disclosed, e.g., in the series by P.Tijssen, Practice and Theory of Enzyme Immunoassays, 3rd Edition, 1987,Elsevier, N.Y., incorporated by reference herein.

The vaccine composition includes a sufficient amount of the desiredimmunogen, such as the peptides of the disclosure, to elicit an immuneresponse. The amount administered can range from about 0.0001 g/kg toabout 1.0 g/kg, relative to the mass of the animal. Any suitablevertebrate animal is readily employed to obtain polyclonal antiserum.Preferably, the animal is a mammal, and includes, but is not limited to,rodents, such as a mice, rats, rabbits, horses, canines, felines,bovines, ovines, e.g., goats and sheep, primates, e.g., monkeys, greatapes and humans, and the like.

The vaccine composition is readily administered by any standard route,including intravenously, intramuscularly, subcutaneously,intraperitoneally, and/or orally. The artisan will appreciate that thevaccine composition is preferably formulated appropriately for each typeof recipient animal and route of administration.

Other aspects of the disclosure relate to methods of treating orpreventing of Zika virus infection by administering to a subject in needthereof an effective amount of a vaccine according to the disclosure.

The present disclosure is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1 Generation of Anti-Zika Virus EP Antibodies

To generate anti-Zika virus envelope protein (EP) antibodies, theFLEP-region located in the ectodomain II (E-DII) of the envelope proteinwas investigated. To determine which scaffold to employ, the RCSBProtein Data Bank (PDB) was utilized and over 500 antigen-antibodystructural complexes were analyzed for interface formation. Ananti-TDRD3 (Tudor Domain Containing 3) human antibody was identified asa scaffold with promising potential to interact with the FLEP-region ofZika virus. Therefore, this antibody was chosen for optimization togenerate antibodies that target the Zika virus EP. The heavy and lightchain variable regions of the anti-TDRD3 are shown in SEQ ID NOs: 4 and5, respectively.

Antibodies targeting Zika virus EP were designed by computing theepitope-paratope connectivity network, as described in Robinson, L. etal., Cell, Vol. 162: 493-504 (2015). The antibodies were designed totarget residues D98, R99 and W101 within the fusion loop. Briefly, thecrystal structure of the anti-TDRD3 antibody in complex with the FLEPwas used to determine the various inter-residue inter-atomic contactsacross the antigen-antibody interface. The interactions between a CDRresidue and its neighboring epitope residues were rendered in a 2Dnetwork graph to analyze the connectivity network. Mutations in the CDRsand/or framework regions that contributed to more favorable contacts, asevaluated by the structural analysis and connectivity network, wereidentified and various amino acid residues which potentially mediate newor improved contacts were analyzed. The variable regions and CDRsgenerated are shown in Tables 2 and 3 as well as in FIGS. 1A and 1B.

The antibodies were then expressed in Freestyle 293 cells by transienttransfection with polyethyleneimine (PEI) and purified by protein Achromatography. The purified antibodies were quantified by IgG ELISA.Briefly, 96-well plates were coated overnight at 4° C. with appropriateantigen. The plates were washed and blocked with 1% blott (Santa CruzBiotechnologies). Serial dilution of antibodies were added to the plateand incubated for 2 hours at room temperature. Antigen bound IgG wasdetected using RbaHu IgG HRP conjugated secondary antibody (JacksonImmunoResearch) followed by TMB substrate (KPL) addition.

Example 2 Binding Affinity of Zika Virus EP Antibodies

To determine whether the antibodies generated were capable of binding tothe Zika virus, a sandwich ELISA was used. Specifically, mAbs 3, 6, 7,and 8, as shown in Table 2, were tested. In addition, a fusion looptargeting pan-flavivirus antibody (4G2) and HIV-1 neutralizing antibody(PGT124) were utilized. The heavy and light variable region sequencesfor 4G2 are set forth in SEQ ID NOs: 12 and 18, respectively. The heavyand light variable region sequences for PGT124 are set forth in SEQ IDNOs: 13 and 19, respectively. The PGT124 antibody targets N-glycan whichis present near the fusion loop epitope region of the Zika virus.

Microtiter plates were coated with 0.05μg of purified mouse 4G2 incarbonate buffer (pH 9.6) overnight at 4° C. and blocked with 10% BSAfor 2 hours at room temperature. Thereafter, 5×10⁴ pfu of Zika viruswere added. Specifically, the strains H/PF/2013 (French Polynesia 2013),ILM (Brazil Paraiba 2015), or MR766-NIID (Uganda 1947) were utilized.After 1 hour incubation at room temperature, serial two-fold dilutionsof antibodies were added for 1 hour, followed by goat anti-human IgG Fccross-adsorbed HRP-conjugated anti-human IgG for 45 minutes. Antibodybinding was visualized by adding 3,3′,5,5′-tetramethylbenzidenesubstrate and reaction was stopped after 10 minutes with sulphuric acid.In between the different steps, plates were washed twice with PBST(PBS+0.05% Tween). Absorbance was read at 450 nm using a plate reader.The results are shown in FIG. 2 and in Table 1 below.

TABLE 1 Kd μg/mL Zika strain Zika strain Zika strain Antibody H/PF/2013ILM MR766 4G2 10.29 6.405 0.0422  PGT124 No/weak binding 388.2 No/weakbinding mAb 3 7.976 0.8088 0.02479 mAb 6 6.542 3.847 0.02852 mAb 7 6.3415.208 0.02651 mAb 8 22.99 19.32 0.02984

These results indicated the antibodies generated in Example 1 werecapable of binding to various Zika virus strains.

Example 3 Neutralization of Zika Virus Envelope Protein Antibodies

To determine whether the anti-Zika virus antibodies could neutralizeZika virus in vitro, purified mAbs were evaluated for their ability toinhibit plaque formation by Zika virus. The plaque neutralization test(PRNT) was performed as previously described (Robinson, L. et al., Cell,Vol. 162: 493-504, 2015). Briefly, the PRNT was performed on BHK-21cells. Serial two-fold dilution of sera containing mAbs 6 and 8 in RPMImaintenance media (MM) was incubated with 50 pfu of Zika virus (ILMstrain or H/PF/2013 strain) in equal volumes for 1 hour before adding toBHK-21. After 1 hour incubation at 37° C., media was aspirated and cellswere overlaid with 1% methyl cellulose in MM. After 5 days at 37° C.,cells were fixed with 20% formaldehyde and stained with 1% crystalviolet. PRNT50 values were determined using a sigmoid dose-responsecurve fit and reported as reciprocal values.

As shown in FIGS. 3A and 3B, mAbs 6 and 8 were capable of neutralizingZika virus. The PRNT50 of mAb 8 was 5.122 μg/mL whereas the PRNT50 ofmAb 6 was 0.0597 g/mL for the ILM strain. The PRNT50 of mAb 8 was 5.092gg/mL for the H/PF/2013 strain. These results indicated that antibodiesgenerated against Zika virus could bind and neutralize the virus.

Example 4 In Vivo Prophylactic and Therapeutic Study

To determine whether antibodies capable of binding and neutralizing Zikavirus in vitro had protective and therapeutic efficacy in vivo, mAbs 6and 8 were tested in adult A129 mice (8-11 weeks old). A129 mice wereinfected intraperitoneally (ip) with H/PF/2013 strain (French Polynesia)at 10³ pfu. To assess protective or prophylactic efficacy of mAbs, micewere injected ip with mAbs (50 pg) one day prior to infection. To assesstherapeutic efficacy, mice were injected ip with mAbs (50 pg) one dayafter infection. Efficacy of mAb was monitored by assessing mortality,weight loss and viremia reduction. In short, mouse blood was collectedfrom facial vein on days 1-8 post-infection to measure serum viremialevel by real-time PCR. Weight was monitored daily until the micesuccumbed to infection. Mouse survival was monitored until 20 dayspost-infection.

As shown in FIGS. 4C and 4D, virus infection caused 100% mortality byday 10 post-infection. In addition, the mice had symptoms ofneuro-related disease (paralysis; data not shown). Treatment with mAb 6or 8 significantly reduced the mortality rate and the loss in bodyweight in both prophylactic and treatment models (FIGS. 4A-4D). Inaddition, FIG. 5 shows mAbs 6 and 8 were able to reduce viremia in miceadministered the antibody either prophylactically or therapeutically.Specifically, viral load was reduced by about 2-3 logs and delayed peakviremia.

The results indicated the anti-Zika virus antibodies generated had bothprophylactic and therapeutic effects.

Example 5 In Vivo Dosage and Antibody-Dependent Enhancement Study

The effect of dosing of anti-Zika virus antibodies on weight loss,viremia and survival was evaluated. A129 mice were administered varyingdoses of mAb 8 (10 mg/kg, 2 mg/kg or 0.2 mg/kg) a day after infectionwith Zika virus (strain H/PF/2013). FIGS. 6A-6C show that at 2 mg/kg(˜60 μg), mAb 8 reduced viremia and provided complete protection againstweight loss and survival. Remarkably, there were no observabledifferences in viremia or accelerated death even when significantlylower doses (0.2 mg/kg˜⁶ μg) of mAb 8 were administered. These resultsindicated that mAb 8 has the potential to provide partial protection,even at significantly low concentrations without increasing viremiaassociated with disease progression.

In addition, antibody-dependent enhancement (ADE) activity of anti-Zikavirus mAb 8 was analyzed. ADE is a phenomenon that has been proposed tomediate increased disease severity when infection occurs in a backgroundof preexisting enhancing antibodies.

Mechanistically, this occurs when enhancing antibodies bind the matureas well as immature virus particles and mediate virus entry via antibodyengagement of Fcγ receptors present on host cells. Several FLE-directedantibodies have been described in literature including 4G2, E53, and theE-dimer epitope (EDE) directed mAbs (Dejnirattisai W, et al., 2015).Several studies have shown that when DENV is opsonized with antibodylevels that are phagocytosed by Fcγ receptors, only antibodies that areable to inhibit virus fusion with phagosomal membranes will preventinfection and thus ADE (Chan K R et al., 2011, Wu R et al, 2012).Accordingly, the ability of mAb8's engagement of the Zika virus FLEepitope at the E-dimer interface to reduce its ADE activity by fusioninhibition was tested.

Specifically, THP1.2S monocyte cells were utilized, which are known toexpress LILRB1 and are thus highly susceptible to ADE. An early designvariant of mAb 8 which showed very weak binding and neutralization ofZIKV H/PF/2013 strain (Kd=50.85 ug/ml; PRNT50>500 ug/ml), was used as acontrol (“mAb control”). Different concentrations of antibody wereincubated with ZIKV (strain H/PF/2013) for 1 hour prior to infectingTHP1.2S monocytes. Seventy hours later the virus replication in culturesupernatants was measured by plaque assay on BHK21 cells. As shown inFIG. 7A, all mAbs tested showed ADE in THP1.2S cells but mAb13 exhibitedhigh ADE activity without any ZIKV neutralization activity despite highantibody concentrations. In contrast, mAb8 showed ADE of at least 3 foldlower viral titers at peak enhancement comparable with 4G2, which isanother fusion loop antibody but which has no neutralization activityagainst ZIKV (FIG. 7B). These results indicated the anti-Zika virus EPantibodies generated in Example 1 were capable of reducing ADE.

Example 6 Efficacy on Maternal Transfer

Placental and fetal infection, along with fetal mortality, has beenobserved in pregnant A129 mice infected with ZIKV. Accordingly, thepotential of mAb 8 to prevent vertical infection and fetal mortality inpregnant A129 mice was evaluated. The overall study design is providedin FIG. 8A. Specifically, 18 pregnant A129 mice that were mated to malemice for 4 days (starting on day 0 evening and separated on day 4morning), were infected with 10³ PFU of Zika virus (H/PF/2013 strain ofAsian lineage) intravenously on day 10 (corresponding to embryo day7-10, i.e., E7-E10). The mice were then treated with 50 μg of mAb 8(n=9) or an isotype control IgG (n=9) for 24 hours (E8-E11) afterinfection. Mice were sacrificed on day 17 (E14-E17) and viral RNA levelswere analyzed on day 12 (blood) and day 17 (blood, placenta and fetalcompartments).

The results of the study are provided in FIGS. 8B-8H. When compared withisotype control IgG treated mice, the mAb 8 treated mice hassubstantially lower levels of viral RNA in blood on day 12 (p-value<0.0001; two-sided t-test) and day 17 (p-value=0.018; two-sided t-test)(FIGS. 8B and 8C). Significantly, mAb 8 treatment reduced placental(p-value <0.0001; two sided t-test) and fetal (p-value <0.0001;two-sided t-test) infection and provided protection against fetalmortality. In contrast, fetuses harvested from the control group had100% lethality, with significant viremia in fetal and placentalcompartments (FIGS. 8D-8F). Fetuses harvested from mice treated with mAb8 showed signs of normal embryo development without any developmentalimpairment. In contrast, fetuses from isotype control IgG treated micewere dead before harvesting, revealing a stark morphological differencecompared with normal fetuses (data not shown). These results indicatedthe anti-Zika virus antibodies described herein are capable ofpreventing vertical infection and fetal mortality in pregnant mice.

Example 7 In Vivo Non-Human Primate Study

Cynomolgus macaques are used to test whether and how effectively ananti-EP mAb can improve survival when administered after high-dose ZIKVinfection.

Zika virus (ZIKV) is produced on Vero cells in complete minimalessential medium (cMEM), 2% FBS, and 1% penicillin/stretopmycin.

Macaques are randomized into groups on the basis of treatment regimens,plus one receiving only PBS as a positive control for infection. Eachsubject is infected with 1000 PFU (1 mL each into two sitesintramuscularly) of ZIKV in Dulbecco's modified Eagle's medium (DMEM).Half of the groups begin treatment 24 hours post infection and the otherhalf of the groups begin treatment 48 hours post infection. The subjectsare treated intravenously with a mAb (25 mg/kg), one of theZIKV-EP-specific neutralizing antibodies disclosed herein, as a 5 mLslow bolus in the saphenous vein. The subjects are monitored daily andscored for disease progression with an internal scoring protocol.Scoring rates changes in the subject's posture/activity, attitude,activity level, feces/urine output, food/water intake, weight,temperature, respiration, and scored disease manifestations such as avisible rash, hemorrhage, cyanosis, or flushed skin. Tests for weight,temperature, blood, and oropharyngeal, nasal, and rectal swabs are takenat days 1, 4, 7, 14, 21, and 28 post infections for the 24-hour group orat 2, 5, 8, 14, 21, and 28 days post infection for the 48-hour group,before the animals receive the mAb.

Example 8 Human Study

Humans infected with Zika virus are given a single anti-EP antibodydisclosed herein. Ideally, the antibodies will be given 24 or 48 hourspost infection. Subjects will be monitored for disease manifestationssuch as visible rash, fevers, or joint pain. In addition, newborns frompregnant women receiving an anti-EP antibody disclosed herein, will bemonitored for microcephaly. Viral titers will also be monitored.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the following claims.

TABLE 2 Antibody Pairs by SEQ ID Number V_(H) CDR V_(L) CDR AntibodyV_(H) V_(L) CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 mAb 1 4 14 20 26 31 38 44 50mAb 2 4 15 20 26 31 38 44 50 mAb 3 9 16 22 28 33 39 45 51 mAb 4 4 16 2026 31 39 45 51 mAb 5 4 17 20 26 31 40 46 50 mAb 6 8 14 21 28 32 38 44 50mAb 7 7 17 21 27 32 40 46 50 mAb 8 6 15 21 27 32 38 44 50 mAb 9 6 5 2127 32 37 43 49 mAb 10 7 5 21 27 32 37 43 49 mAb 11 8 5 21 28 32 37 43 49mAb 12 9 5 22 28 33 37 43 49 mAb 13 10 5 23 28 34 37 43 49 mAb 14 11 521 28 34 37 43 49 mAb 15 6 14 21 27 32 38 44 50 mAb 16 6 16 21 27 32 3945 51 mAb 17 6 17 21 27 32 40 46 50 mAb 18 7 14 21 27 32 38 44 50 mAb 197 15 21 27 32 38 44 50 mAb 20 7 16 21 27 32 39 45 51 mAb 21 8 15 21 2832 38 44 50 mAb 22 8 16 21 28 32 39 45 51 mAb 23 8 17 21 28 32 40 46 50mAb 24 9 14 22 28 33 38 44 50 mAb 25 9 15 22 28 33 38 44 50 mAb 26 9 1722 28 33 40 46 50 mAb 27 10 14 23 28 34 38 44 50 mAb 28 10 15 23 28 3438 44 50 mAb 29 10 16 23 28 34 39 45 51 mAb 30 10 17 23 28 34 40 46 50mAb 31 11 14 21 28 34 38 44 50 mAb 32 11 15 21 28 34 38 44 50 mAb 33 1116 21 28 34 39 45 51 mAb 34 11 17 21 28 34 40 46 50

TABLE 3 Summary Sequence Table SEQ ID NO Description Sequence  1Human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS Heavy ChainGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 Human IgG2RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV Light ChainDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA (kappa)CEVTHQGLSSPVTKSFNRGEC  3 Zika virus DRGWGNGCGLFG fusion proteinloop (residues 98-109 of E- DII)  4 anti-TDRD3EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSYMHWVRQAPG V_(H) Wild TypeKGLEWVASISSSYGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARTVRGSKKPYFSGWAMDYWGQGTLVTVSS  5 anti-TDRD3DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGK V_(L) Wild TypeAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHGPFYW-LFTFGQGTKVEIK  6V_(H).1 EVQLLESGGGLVQPGGSLRLSCAASGFSFSTY SMHWVRQAPG KGLEWVSAISGEGDSAYYADSVKGRFTISRDNSKNTLYLQMN KVRAEDTAVYYCV----G GYSNFYYYYTMDA WGQGTMVTVSS(V5L, N285, L29F, S31T, S32Y, Y335, A495, S50A, S52(A)G,S53E, Y54G, G55D, T57A, A71R, T73N, A78L, S82(B)K, L82(C)V,A93V, S99G, K100Y, K100(A)S, P100(B)N, Y100(C)F, F100(D)Y,S100(E)Y, G100(F)Y, W100(G)Y, A100(H)T, Y102(A), L108M)  7 V_(H).2EVQLLESGGGLVQPGGSLRLSCAASGFSFSTY SMHWVRQAPG KGLEWVSAISGEGDSAYYADSVKGRFEISRDNSKNTLYLQMN KVRAEDTAVYYCV----G GYSNFYYYYTMDA WGQGTMVTVSS(V5L, N285, L29F, S31T, S32Y, Y335, A495, S50A, S52(A)G,

L82(C)V, A93V, S99G, K100Y, K100(A)S, P100(B)N, Y100(C)F,F100(D)Y, S100(E)Y, G100(F)Y, W100(G)Y, A100(H)T, Y102A, L108M)  8V_(H).3 EVQLVESGGGLVQPGGSLRLSCSASGFSFSTY SMHWVRQAPG KGLEYVSAIT GEGDSAFYADSVKGRFTISRDNSKNTLYFEMNS LRPEDTAVYYCV----G GYSNFYYYYTMDA WGQGTSVTVSS

Y100(C)F, F100(D)Y, S100(E)Y, G100(F)Y, W100(G)Y, A100(H)T,

 9 V_(H).4 EVQLVESGGGLVQPGGSLRLSCSASGFTFSTY SMHWVRQAPG KGLEYVSAI TGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNS LRPEDTAVYYCV----G GYSNFYYYYTMDV WGQGTTVTVSS

S52(A)G, S53E, Y54G, G55D, T57A, Y58F, A71R, T73N, A78L,L80F, Q81E, A84P, A93V, S99G, K100Y, K100(A)S, P100(B)N,Y100(C)F, F100(D)Y, S100(E)Y, G100(F)Y, W100(G)Y, A100(H)T,

10 V_(H).5 QVQLVESGGGLVQPGGSLRLSCSASGFFSTY SMHWVKQAPGK GLEYVSAI TGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNSL RPEDTAVYYCV----G GYTNFYYYYTMDA WGQGTSVTVSS

S52T, S52(A)G, S53E, Y54G, G55D, T57A, Y58F, A71R, T73N,

P100(B)N, Y100(C)F, F100(D)Y, S100(E)Y, G100(F)Y, W100(G)Y,

11 V_(H).6 QVQLVESGGGLVQPGGSLRLSCSASGFSFSTY SMHWVKQAPG KGLEYVSAI TGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNS LRPEDTAVYYCV----G GYTNFYYYYTMDA WGQGTSVTVSS(EQ1, A23S, N28S, L29F, S31T, S32Y, Y33S, R38K, W47Y, A49S,S50A, S52T, S52(A)G, S53E, Y54G, G55D, T57A, Y58F, A71R,T73N, A78L, L80F, Q81E, A84P, A93V, S99G, K100Y, K100(A)T,P100(B)N, Y100(C)F, F100(D)Y, S100(E)Y, G100(F)Y, W100(G)Y,A100(H)T, Y102A, L108S) 12 V_(H) of fusionEVQLQQSGPELVKPGTSVKISCKTSGYTFTEYTIHWVKQSHGK loop targetingSLAWIGGIDPNSGGTNYSPNFKGKATLTVDKSSSTAYMDLRSL pan-flavivirusSSEDSAVYFCARIYHYDGYFDVWGAGTAVTVSS antibody 4g2 13 V_(H) of anti-QVQLQESGPGLVRPSETLSVTCIVSGGSISNYYWTWIRQSPGKG HIVLEWIGYISDRETTTYNPSLNSRAVISRDTSKNQLSLQLRSVTTA neutralizingDTAIYFCATARRGQRIYGVVSFGEFFYYYYMDVWG antibody KGTAVTVSS PGT124 14 V_(L).1EIVLTQSPASLSLSPGERATLSCRATOSISTFLAWYQHKPGQA PRLLIYDASTRASGVPARFSGSRSGTDFTLTISSLEPEDFAVYYC QQR-YNWPPYS FGQGTKVEIK(D1E, Q3V, M4L, 59A, A13L, V15P, D17E, V19A, I21L, Y225,S26T, V29I, S31T, A32F, V33L, Q38H, K42Q, K45R, S50D, S53T,L54R, Y55A, S60A, Q79E, T85V, H91R, F94Y, Y95N, L95(b)P, F96Y, T975) 15V_(L).2 DIVMTQSPASLSLSPGERATLSCRATOSISTFLAWYQQKPGQ APRLLIYDASTRASGIPARFSGSRSGTDFTLTITRLEPEDFAVYY COOR-YNWPPYS FGQGTKLEIK(Q3V, S9A, A13L, V15P, D17E, V19A, I21L, Y22S, S26T, V29I,

T97S, V104L) 16 V_(L).3 EIVLTQSPATLSLSPGERATLSCRASOSISTFLAWYQHKPGQAPRLLIY DASTRAT GVPARFSGSRSGTDFTLTISTLEPEDFAVYY CQQR-YNWPPYTFGQGTKVEIK

Y22S, V29I, S31T, A32F, V33L, Q38H, K42Q, K45R, S50D, S53T,

L95(b)P, F96Y) 17 V_(L).4 DIVMTQSPASLSLSPGERATLSCRATQSIVTFLAWYQQKPGQAPRLLIY DASTNASGIPARFSGSRSGTDFTLTITRLEPEDFAVYY CQQR-YNWPPYS FGQGTKLEIK(Q3V, S9A, A13L, V15P, D17E, V19A, I21L, Y22S, S26T, V29I,

F96Y, T97S, V104L) 18 V_(L) of fusionDIKMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKS loop targetingPKTLIYRANRLIDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYY pan-flavivirusCLQYDEFPPTFGGGTKLEIKR antibody 4g2 19 V_(L) of anti-SYVSPLSVALGETARISCGRQALGSRAVQWYQHKPGQAPILLI HIVYNNQDRPSGIPERFSGTPDINFGTTATLTISGVEVGDEADYYCH neutralizingMWDSRSGFSWSFGGATRLTVLSQP antibody PGT124 20 anti-TDRD3 GFNLSSSV_(H) CDR1 21 V_(H).1CDR1 GFSFSTY 22 V_(H).4CDR1 GFTFSTY 23 V_(H).5CDR1GF-FSTY 24 4g2 V_(H) CDR1 GYTFTEY 25 PGT124 V_(H) GGSISNY CDR1 26anti-TDRD3 SSSYGS V_(H) CDR2 27 V_(H).1 CDR2 SGEGDS 28 V_(H).3 CDR2TGEGDS 29 4g2 V_(H) CDR2 DPNSGG 30 PGT124 V_(H) SDRET CDR2 31 anti-TDRD3TVRGSKKPYFSGWAMDY V_(H) CDR3 32 V_(H).1CDR3 ----GYSNFYYYYTMDA 33V_(H).4CDR3 ----GYSNFYYYYTMDV 34 V_(H).5CDR3 ----GYTNFYYYYTMDA 354g2 V_(H) CDR3 IYHYDGYFDV 36 PGT124 V_(H) ARRGQRIYGVVSFGEFFYYYYMDV CDR337 anti-TDRD3 RASQSVSSAVA V_(L)CDR1 38 V_(L).1CDR1 RATQSISTFLA 39V_(L).3CDR1 RASQSISTFLA 40 V_(L).4CDR1 RATQSIVTFLA 41 4g2 V_(L) CDR1KASQDINSYLT 42 PGT124 V_(L) GRQALGSRAVQ CDR1 43 anti-TDRD3 SASSLYSV_(L) CDR2 44 V_(L).1CDR2 DASTRAS 45 V_(L).3CDR2 DASTRAT 46 V_(L).4CDR2DASTNAS 47 4g2 V_(L) CDR2 RANRLID 48 PGT124 V_(L) NNQDRPS CDR2 49anti-TDRD3 QQHGPFYWLFT V_(L) CDR3 50 V_(L).1CDR3 QQR--YNWPPYS 51V_(L).3CDR3 QQR--YNWPPYT 52 4g2 V_(L) CDR3 LQYDEFPPT 53 PGT124 V_(L)HMWDSRSGFSWS CDR3 54 V_(H) CDR1 GFX₁FSTY 55 V_(H) CDR2 X₂GEGDS 56V_(H) CDR3 GYX₃NFYYYYTMDX₄ 57 V_(L) CDR1 RAX₅QSIX₆TFLA 58 V_(L) CDR2DASTX₇AX₈ 59 V_(L) CDR3 QQRYNWPPYX₉

1. An isolated monoclonal antibody which specifically binds Zika virus envelope protein, or antigen binding portion thereof, comprising heavy and light chain CDRs, wherein (i) heavy chain CDR1 comprises GFX₁FSTY, wherein X₁ may or may not be present, and if present is a polar amino acid residue; (ii) heavy chain CDR2 comprises X₂GEGDS, wherein X₂ is a polar amino acid residue; (iii) heavy chain CDR3 comprises GYX₃NFYYYYTMDX₄, wherein X₃ is a polar amino acid residue and X₄ is a nonpolar amino acid residue; (iv) light chain CDR1 comprises RAX₅QSIX₆TFLA, wherein X₅ is a polar amino acid residue and X₆ is a polar amino acid residue or a hydrophobic amino acid residue; (v) light chain CDR2 comprises DASTX₇AX₈, wherein X₇ and X₈ are polar amino acids; and (vi) light chain CDR3 comprises QQRYNWPPYX₉, wherein X₉ is a polar amino acid.
 2. The isolated monoclonal antibody, or antigen binding portion thereof of claim 1, wherein (i) heavy chain CDR1 comprises GFX₁FSTY, wherein X₁ is selected from S and T; (ii) heavy chain CDR2 comprises X₂GEGDS, wherein X₂ is selected from S and T; (iii) heavy chain CDR3 comprises GYX₃NFYYYYTMDX₄, wherein X₃ is selected from S and T and X₄ is selected from A and V; (iv) light chain CDR1 comprises RAX₅QSIX₆TFLA, wherein X₅ is selected from S and T and X₆ is selected from S and V; (v) light chain CDR2 comprises DASTX₇AX₈, wherein X₇ is selected from R and N and X₈ is selected from S and T; and (vi) light chain CDR3 comprises QQRYNWPPYX₉, wherein X₉ is selected from S and T.
 3. The isolated monoclonal antibody, or antigen binding portion thereof of claim 1, wherein (i) heavy chain CDR1 comprises GFX₁FSTY, wherein X₁ is not present; (ii) heavy chain CDR2 comprises X₂GEGDS, wherein X₂ is selected from S and T; (iii) heavy chain CDR3 comprises GYX₃NFYYYYTMDX₄, wherein X₃ is selected from S and T and X₄ is selected from A and V; (iv) light chain CDR1 comprises RAX₅QSIX₆TFLA, wherein X₅ is selected from S and T and X_(b) is selected from S and V; (v) light chain CDR2 comprises DASTX₇AX₈, wherein X₇ is selected from R and N and X₈ is selected from S and T; and (vi) light chain CDR3 comprises QQRYNWPPYX₉, wherein X₉ is selected from S and T.
 4. The isolated monoclonal antibody, or antigen binding portion thereof of claim 1, wherein (i) heavy chain CDR1 comprises GFSFSTY; (ii) heavy chain CDR2 comprises SGEGDS; and (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA; or (i) heavy chain CDR1 comprises GFSFSTY; (ii) heavy chain CDR2 comprises TGEGDS; and (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA; or (i) heavy chain CDR1 comprises GFTFSTY; (ii) heavy chain CDR2 comprises TGEGDS; and (iii) heavy chain CDR3 comprises GYSNFYYYYTMDV; or (i) heavy chain CDR1 comprises GFFSTY; (ii) heavy chain CDR2 comprises TGEGDS; and (iii) heavy chain CDR3 comprises GYTNFYYYYTMDA; or (i) heavy chain CDR1 comprises GFSFSTY; (ii) heavy chain CDR2 comprises TGEGDS; and (iii) heavy chain CDR3 comprises GYTNFYYYYTMDA. 5.-8. (canceled)
 9. The isolated monoclonal antibody, or antigen binding portion thereof of claim 1, wherein (iv) light chain CDR1 comprises RATQSISTFLA; (v) light chain CDR2 comprises DASTRAS; and (vi) light chain CDR3 comprises QQRYNWPPYS; or (iv) light chain CDR1 comprises RASQSISTFLA; (v) light chain CDR2 comprises DASTRAT; and (vi) light chain CDR3 comprises QQRYNWPPYT; or (iv) light chain CDR1 comprises RATQSIVTFLA; (v) light chain CDR2 comprises DASTNAS; and (vi) light chain CDR3 comprises QQRYNWPPYS. 10.-11. (canceled)
 12. The isolated monoclonal antibody, or antigen binding portion thereof of claim 1, wherein (i) heavy chain CDR1 comprises GFSFSTY; (ii) heavy chain CDR2 comprises SGEGDS; (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA; (iv) light chain CDR1 comprises RATQSISTFLA; (v) light chain CDR2 comprises DASTRAS; and (vi) light chain CDR3 comprises QQRYNWPPYS; or (i) heavy chain CDR1 comprises GFSFSTY; (ii) heavy chain CDR2 comprises SGEGDS; (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA; (iv) light chain CDR1 comprises RATQSIVTFLA; (v) light chain CDR2 comprises DASTNAS; and (vi) light chain CDR3 comprises QQRYNWPPYS; or (i) heavy chain CDR1 comprises GFSFSTY; (ii) heavy chain CDR2 comprises TGEGDS; (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA; (iv) light chain CDR1 comprises RATQSISTFLA; (v) light chain CDR2 comprises DASTRAS; and (vi) light chain CDR3 comprises QQRYNWPPYS; or (i) heavy chain CDR1 comprises GFTFSTY; (ii) heavy chain CDR2 comprises TGEGDS; (iii) heavy chain CDR3 comprises GYSNFYYYYTMDV; (iv) light chain CDR1 comprises RASQSISTFLA; (v) light chain CDR2 comprises DASTRAT; and (vi) light chain CDR3 comprises QQRYNWPPYT. 13.-15. (canceled)
 16. An isolated monoclonal antibody which specifically binds Zika virus envelope protein, or antigen binding portion thereof, comprising a heavy chain variable region as set forth in SEQ ID NO: 4, wherein (i) heavy chain CDR1 comprises an amino acid substitution or deletion at N28, and amino acid substitutions at L29, S31, S32; (ii) heavy chain CDR2 comprises amino acid substitutions at S52, S52A, S53, Y54, G55; (iii) heavy chain CDR3 comprises an amino acid deletion at S99 and amino acid substitutions at K100, K100A, P100B, Y100C, F100D, S100E, G100F, W100Q A100H, and Y102; and wherein the heavy chain variable region comprises at least one amino acid deletion at R94, and at CDR3 residues T95, V96, and R97, and combinations thereof, numbering according to Chothia.
 17. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein the heavy chain variable region further comprises at least one amino acid substitution at V5, Y33, A49, S50, T57, A71, T73, A78, S82B, L82C, A93, L108 and combinations thereof, numbering according to Chothia.
 18. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein the heavy chain variable region further comprises an amino acid substitution at T68, numbering according to Chothia.
 19. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein the heavy chain variable region further comprises at least one amino acid substitution at A23, W47, Y58, L80, Q81, A84, and combinations thereof, numbering according to Chothia.
 20. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein the heavy chain variable region further comprises at least one amino acid substitution at E1, A23, R38, W47, Y58, L80, Q81, A84, and combinations thereof, numbering according to Chothia.
 21. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein the heavy chain variable region further comprises at least one amino acid substitution at E1, A23, R38, W47, Y58, T68, L80, Q81, A84, and combinations thereof, numbering according to Chothia.
 22. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein (i) heavy chain CDR1 comprises N28S or N28T, L29F, S31T, S32Y; (ii) heavy chain CDR2 comprises S52T, S52AG, S53E, Y54G, G55D; (iii) heavy chain CDR3 comprises K100Y, K100AS or K100AT, P100BN, Y100CF, F100DY, S100EY, G100FY, W100GY, A100HT, and Y102A or Y102V.
 23. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein the heavy chain variable region comprises amino acid deletions at R94, and at CDR3 residues T95, V96, and R97, numbering according to Chothia.
 24. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 5, wherein (i) light chain CDR1 comprises amino acid substitutions at S26, V29, S31, A32, and V33; (ii) light chain CDR2 comprises amino acid substitutions at S50, S53, L54, Y55 and optionally S56; and (iii) light chain CDR3 comprises amino acid substitutions at H91, P93, F94, Y95, L95B, F96, and T97 and an amino acid deletion at G92.
 25. The isolated monoclonal antibody, or antigen binding portion thereof of claim 24, wherein (i) light chain CDR1 comprises S26T, V29I, S31V, A32F, and V33L; (ii) light chain CDR2 comprises S50D, S53T, L54R or L54N, Y55A and, optionally S56T; and (iii) light chain CDR3 comprises amino acid substitutions at H91R, P93Y, F94N, Y95W, L95BP, F96Y, and T97S.
 26. The isolated monoclonal antibody, or antigen binding portion thereof of claim 16, wherein the light chain variable region further comprises at least one amino acid substitution at D1, Q3, M4, S9, A13, V15, D17, V19, I21, Y22, Q38, K42, K45, S60, Q79, T85, and combinations thereof, numbering according to Chothia.
 27. The isolated monoclonal antibody, or antigen binding portion thereof of claim 26, wherein the light chain variable region further comprises at least one amino acid substitution at S10, V58, S76, S77, V104, and combinations thereof, numbering according to Chothia.
 28. An isolated monoclonal antibody, or antigen binding portion thereof, which binds to Zika virus envelope protein and comprises heavy and light chain variable regions, wherein the heavy and light chain amino acid sequences are selected from the group consisting of: (a) SEQ ID NOs: 4 and 14, respectively; (b) SEQ ID NOs: 4 and 15, respectively; (c) SEQ ID NOs: 9 and 16, respectively; (d) SEQ ID NOs: 4 and 16, respectively; (e) SEQ ID NOs: 4 and 17, respectively; (f) SEQ ID NOs: 8 and 14, respectively; (g) SEQ ID NOs: 7 and 17, respectively; (h) SEQ ID NOs: 6 and 15, respectively; (i) SEQ ID NOs: 6 and 5, respectively; (j) SEQ ID NOs: 7 and 5, respectively; (k) SEQ ID NOs: 8 and 5, respectively; (l) SEQ ID NOs: 9 and 5, respectively; (m) SEQ ID NOs: 10 and 5, respectively; (n) SEQ ID NOs: 11 and 5, respectively; (o) SEQ ID NOs: 6 and 14, respectively; (p) SEQ ID NOs: 6 and 16, respectively; (q) SEQ ID NOs: 6 and 17, respectively; (r) SEQ ID NOs: 7 and 14, respectively; (s) SEQ ID NOs: 7 and 15, respectively; (t) SEQ ID NOs: 7 and 16, respectively; (u) SEQ ID NOs: 8 and 15, respectively; (v) SEQ ID NOs: 8 and 16, respectively; (w) SEQ ID NOs: 8 and 17, respectively; (x) SEQ ID NOs: 9 and 14, respectively; (y) SEQ ID NOs: 9 and 15, respectively; (z) SEQ ID NOs: 9 and 17, respectively; (aa) SEQ ID NOs: 10 and 14, respectively; (bb) SEQ ID NOs: 10 and 15, respectively; (cc) SEQ ID NOs: 10 and 16, respectively; (dd) SEQ ID NOs: 10 and 17, respectively; (ee) SEQ ID NOs: 11 and 14, respectively; (ff) SEQ ID NOs: 11 and 15, respectively; (gg) SEQ ID NOs: 11 and 16, respectively; and (hh) SEQ ID NOs: 11 and 17, respectively.
 29. An isolated monoclonal antibody, or antigen binding portion thereof, which binds to Zika virus envelope protein, comprising heavy and light chain CDRs selected from the group consisting of: (a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 20, 26 and 31, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively; (b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 22, 28 and 33, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively; (c) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 20, 26 and 31, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively; (d) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 20, 26 and 31, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively; (e) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 28 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively; (f) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 27 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively; (g) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 27 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively; (h) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 27 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively; (i) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 28 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively; (j) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 22, 28 and 33, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively; (k) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 23, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively; (l) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49, respectively; (m) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 27 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively; (n) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 28 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively; (o) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 28 and 32, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively; (p) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 22, 28 and 33, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively; (q) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 22, 28 and 33, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively; (r) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 23, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively; (s) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 23, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively; (t) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 23, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively; (u) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50, respectively; (v) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51, respectively; and (w) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 21, 28 and 34, respectively, and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50, respectively.
 30. An isolated monoclonal antibody, or antigen binding portion thereof, which binds to Zika virus envelope protein and comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 7, 8, 9, 10 and 11; and wherein the light chain variable region comprises an amino acid sequence which is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 14, 15, 16, and 17, provided that the monoclonal antibody does not comprise SEQ ID NOs: 4 and
 5. 31. The isolated monoclonal antibody, or antigen binding portion thereof, according to claim 30, comprising heavy chain and light chain sequences having at least 90% identity to the heavy and light chain amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 4 and 14, respectively; (b) SEQ ID NOs: 4 and 15, respectively; (c) SEQ ID NOs: 9 and 16, respectively; (d) SEQ ID NOs: 4 and 16, respectively; (e) SEQ ID NOs: 4 and 17, respectively; (f) SEQ ID NOs: 8 and 14, respectively; (g) SEQ ID NOs: 7 and 17, respectively; (h) SEQ ID NOs: 6 and 15, respectively; (i) SEQ ID NOs: 6 and 5, respectively; (j) SEQ ID NOs: 7 and 5, respectively; (k) SEQ ID NOs: 8 and 5, respectively; (l) SEQ ID NOs: 9 and 5, respectively; (m) SEQ ID NOs: 10 and 5, respectively; (n) SEQ ID NOs: 11 and 5, respectively; (o) SEQ ID NOs: 6 and 14, respectively; (p) SEQ ID NOs: 6 and 16, respectively; (q) SEQ ID NOs: 6 and 17, respectively; (r) SEQ ID NOs: 7 and 14, respectively; (s) SEQ ID NOs: 7 and 15, respectively; (t) SEQ ID NOs: 7 and 16, respectively; (u) SEQ ID NOs: 8 and 15, respectively; (v) SEQ ID NOs: 8 and 16, respectively; (w) SEQ ID NOs: 8 and 17, respectively; (x) SEQ ID NOs: 9 and 14, respectively; (y) SEQ ID NOs: 9 and 15, respectively; (z) SEQ ID NOs: 9 and 17, respectively; (aa) SEQ ID NOs: 10 and 14, respectively; (bb) SEQ ID NOs: 10 and 15, respectively; (cc) SEQ ID NOs: 10 and 16, respectively; (dd) SEQ ID NOs: 10 and 17, respectively; (ee) SEQ ID NOs: 11 and 14, respectively; (ff) SEQ ID NOs: 11 and 15, respectively; (gg) SEQ ID NOs: 11 and 16, respectively; and (hh) SEQ ID NOs: 11 and 17, respectively.
 32. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, having neutralizing activity against Zika virus.
 33. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, wherein the antibody is selected from the group consisting of an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, an IgD, and an IgE antibody.
 34. The isolated monoclonal antibody, or antigen binding portion thereof, according to claim 33, wherein the antibody is an IgG1 antibody.
 35. A pharmaceutical composition comprising an isolated monoclonal antibody or antigen binding portion thereof, of claim 1, and a pharmaceutically acceptable carrier.
 36. A method for treating Zika virus infection comprising administering to a subject in need thereof, an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 37. A nucleic acid comprising a nucleotide sequence encoding the light chain, heavy chain, or both light and heavy chains of the isolated monoclonal antibody or antigen binding portion thereof, of claim
 1. 38. An expression vector comprising the nucleic acid of claim
 37. 39. A cell transformed with an expression vector of claim
 38. 40. A method for preventing Zika virus infection in a subject, comprising administering to the subject in need thereof, an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 41. A method for treating or preventing vertical Zika virus infection to a fetus in a pregnant subject, comprising administering to the subject an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 42. The method of claim 41, wherein the pregnant subject is infected with a Zika virus or the pregnant subject is at risk of Zika virus infection.
 43. (canceled)
 44. A method for treating or preventing fetal Zika virus infection, comprising administering to a pregnant subject in need thereof, an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 45. The method of claim 44, wherein the pregnant subject is infected with Zika virus or the pregnant subject is at risk of being infected with Zika virus.
 46. (canceled)
 47. A method for treating or preventing fetal mortality in a pregnant subject, comprising administering to the subject an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 48. The method of claim 47, wherein the pregnant subject is infected with Zika virus or the pregnant subject is at risk of being infected with Zika virus.
 49. (canceled)
 50. A method for treating or preventing placental Zika virus infection, comprising administering to a pregnant subject in need thereof, an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 51. The method of claim 50, wherein the pregnant subject is infected with Zika virus or the pregnant subject is at risk of being infected with Zika virus.
 52. (canceled)
 53. A method for reducing or reducing the risk of fetal mortality in a pregnant subject, comprising administering to the subject an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 54. The method of claim 53, wherein the pregnant subject is infected with Zika virus or the pregnant subject is at risk of being infected with Zika virus.
 55. (canceled)
 56. A method for reducing or reducing the risk of fetal Zika virus infection, comprising administering to a pregnant subject in need thereof, an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 57. The method of claim 56, wherein the pregnant subject is infected with Zika virus or the pregnant subject is at risk of being infected with Zika virus.
 58. (canceled)
 59. A method for reducing or reducing the risk of placental Zika virus infection, comprising administering to a pregnant subject in need thereof, an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 60. The method of claim 59, wherein the pregnant subject is infected with Zika virus or the pregnant subject is at risk of being infected with Zika virus.
 61. (canceled)
 62. A method for reducing or reducing the risk of vertical Zika virus infection to a fetus in a pregnant subject, comprising administering to the subject an effective amount of an isolated monoclonal antibody, or antigen binding portion thereof, of claim
 1. 63. The method of claim 62, wherein the pregnant subject is infected with a Zika virus or the pregnant subject is at risk of Zika virus infection.
 64. (canceled) 